THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
E. G. CONKLIN, Princeton University
E. N. HARVEY, Princeton University
SELIG HECHT, Columbia University
LEIGH HOADLEY, Harvard University
L. IRVING, Swarthmore College
M. H. JACOBS, University of Pennsylvania
H. S. JENNINGS, Johns Hopkins University
FRANK R. LILLIE, University of Chicago
CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
A. C. REDFIELD, Harvard University
F. SCHRADER, Columbia University
H. B. STEINBACH, Washington University
Managing Editor
VOLUME 85
AUGUST TO DECEMBER, 1943
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE 8C LEMON STS.
LANCASTER, PA.
11
THE BIOLOGICAL BULLETIN is issued six times a year at the
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn-
sylvania.
Subscriptions and similar matter should be addressed to The
Biological Bulletin, Marine Biological Laboratory, Woods Hole,
Massachusetts. Agent for Great Britain: Wheldon and Wesley,
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
W. C. 2. Single numbers, $1.75. Subscription per volume (three
issues), $4.50.
Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Massachusetts, between July 1 and October 1, and to the Depart-
ment of Zoology, Washington University, St. Louis, .Missouri,
during the remainder of the year.
Entered as second-class matter May 17, 1930, at the post office at Lancaster,
Pa., under the Act of August 24, 1912.
LANCASTER PRESS, INC., LANCASTER, PA.
CONTENTS
No. 1. AUGUST, 1943
PAGE
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 1
SONNEBORN, T. M., AND RUTH V. DlPPELL
Sexual Isolation, Mating Types, and Sexual Responses to Diverse Con-
ditions in Variety 4, Paramecium Aurelia 36
HOVANITZ, WILLIAM
Hybridization and Seasonal Segregation in Two Races of a Butterfly
Occurring Together in Two Localities 44
LEVINE, HARRY P.
Species Differences in Rates of Osmotic Hemolysis Within the Genus
Peromyscus 52
LAWSON, CHESTER A.
Germarial Differences and the Production of Aphid Types 60
LOOSANOFF, VICTOR L., AND JAMES B. ENGLE
Polydora in Oysters Suspended in Water 69
WAIT, ROBERT B.
The Action of Acetylcholine on the Isolated Heart of Venus Mercenaria 79
No, 2. OCTOBER, 1943
PYLE, ROBERT W.
The Histogenesis and Cyclic Phenomena of the Sinus Gland and X-
Organ in Crustacea 87
BURT, AGNES SANXAY
Neurulation in Mechanically and Chemically Inhibited Amblystoma. . 103
PRATT, DAVID M.
Analysis of Population Development in Daphnia at Different Tempera-
tures 116
HARVEY, ETHEL BROWNE
Rate of Breaking and Size of the "Halves" of the Arbacia Punctulata
Egg when Centrifuged in Hypo- and Hypertonic Sea Water 141
HARVEY, ETHEL BROWNE, AND THOMAS F. ANDERSON
The Spermatozoon and Fertilization Membrane of Arbacia Punctulata
as Shown by the Electron Microscope 151
BODINE, JOSEPH HALL, AND THEODORE NEWTON TAHMISIAN
The Development of an Enzyme (Tyrosinase) in the Parthenogenetic
Egg of the Grasshopper, Melanoplus Differentialis 157
Ris, HANS
A Quantitative Study of Anaphase Movement in the Aphid Tamalia. . 164
iv CONTENTS
PAGE
No. 3. DECEMBER, 1943
HARRIS, DANIEL L.
The Osmotic Properties of Cytoplasmic Granules of the Sea Urchin Egg 179
WILBUR, KARL M., AND RICHARD O. RECKNAGEL
The Radiosensitivity of Eggs of Arbacia Punctulata in Various Salt
Solutions 193
CLARKE, GEORGE L., E. LOWE PIERCE AND DEAN F. BUMPUS
The Distribution and Reproduction of Sagitta Elegans on Georges Bank
in Relation to the Hydrographical Conditions 201
STUNKARD, HORACE W.
The Morphology and Life History of the Digenetic Trematode, Zoogo-
noides Laevis Linton, 1940 227
WHITING, P. W.
Intersexual Females and Intersexuality in Habrobracon 238
THIVY, FRANCESCA
New Records of Some Marine Chaetophoraceae and Chaetosphaeridia-
ceae for North America t. . . . 244
HUGHES-SCHRADER, SALLY
Polarization, Kinetochore Movements, and Bivalent Structure in the
Meiosis of Male Mantids. 265
Vol. 85, No. 1 August, 1943
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
FORTY-FIFTH REPORT, FOR THE YEAR 1942 — FIFTY-FIFTH YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 11, 1942) .... 1
STANDING COMMITTEES 2
II. ACT OF INCORPORATION 3
III. BY-LAWS OF THE CORPORATION 4
IV. REPORT OF THE TREASURER 5
V. REPORT OF THE LIBRARIAN 9
VI. REPORT OF THE DIRECTOR 11
Statement 11
Addenda :
1. The Staff, 1942 13
2. Investigators and Students, 1942 16
3. Tabular View of Attendance 21
4. Subscribing and Co-operating Institutions, 1942 22
5. Evening Lectures, 1942 22
6. Shorter Scientific Papers, 1942 23
7. Members of the Corporation 24
I. TRUSTEES
EX OFFICIO
FRANK R. LILLIE, President Emeritus of the Corporation, The University of Chicago.
LAWRASON RIGGS, President of the Corporation, 120 Broadway, New York City.
E. NEWTON HARVEY, Vice President of the Corporation, Princeton University.
CHARLES PACKARD, Director, Marine Biological Laboratory.
OTTO C. GLASER, Clerk of the Corporation, Amherst College.
DONALD M. BRODIE, Treasurer, 522 Fifth Avenue, New York City.
EMERITUS
Ross G. HARRISON, Yale University.
H. S. JENNINGS, University of California.
C. E. McCLUNG, University of Pennsylvania.
S. O. MAST, Johns Hopkins University.
A. P. MATHEWS, University of Cincinnati.
T. H. MORGAN, California Institute of Technology.
W. J. V. OSTERHOUT, Rockefeller Institute.
G. H. PARKER, Harvard University.
W. B. SCOTT, Princeton University.
1
MARINE BIOLOGICAL LABORATORY
TO SERVE UNTIL 1946
DUGALD E. S. BROWN, New York University.
E. R. CLARK, University of Pennsylvania.
OTTO C. GLASER, Amherst College.
E. N. HARVEY, Princeton University.
M. H. JACOBS, University of Pennsylvania.
F. P. KNOWLTON, Syracuse University.
FRANZ SCHRADER, Columbia University.
B. H. WILLIER, Johns Hopkins University.
TO SERVE UNTIL 1945
W. R. AMBERSON, University of Maryland School of Medicine.
S. C. BROOKS, University of California.
W. C. CURTIS, University of Missouri.
H. B. GOODRICH, Wesleyan University.
I. F. LEWIS, University of Virginia.
R. S. LILLIE, The University of Chicago.
A. C. REDFIELD, Harvard University.
C. C. SPEIDEL, University of Virginia.
TO SERVE UNTIL 1944
ERIC G. BALL, Harvard University Medical School.
R. CHAMBERS, Washington Square College, New York University.
EUGENE F. DuBois, Cornell University Medical College.
W. E. CARREY, Vanderbilt University Medical School.
COLUMBUS ISELIN, Woods Hole Oceanographic Institution.
C. W. METZ, University of Pennsylvania.
H. H. PLOUGH, Amherst College.
W. R. TAYLOR, University of Michigan.
TO SERVE UNTIL 1943
W. C. ALLEE, The University of Chicago.
G. H. A. CLOWES, Lilly Research Laboratory.
B. M. DUGGAR, University of Wisconsin.
L. V. HEILBRUNN, University of Pennsylvania.
LAURENCE IRVING, Swarthmore College.
J. H. NORTHROP, Rockefeller Institute.
A. H. STURTEVANT, California Institute of Technology.
LORANDE L. WOODRUFF, Yale University.
EXECUTIVE COMMITTEE OF THE BOARD OK TRUSTEES
LAWRASON RIGGS, Ex officio, Chairman.
E. N. HARVEY, Ex officio.
D. M. BRODIE, Ex officio.
CHARLES PACKARD, Ex officio.
D. E. S. BROWN, to serve until 1943.
B. H. WILLIER, to serve until 1943.
C. W. METZ, to serve until 1944.
OTTO C. GLASER, to serve until 1944.
ACT OF INCORPORATION
THE LIBRARY COMMITTEE
A. C. REDFIELD, Chairman.
E. G. BALL.
S. C. BROOKS.
M. E. KRAHL.
J. W. MAYOR.
D. E. S. BROWN, Chairman.
C. L. CLAFF.
G. FAILLA.
S. E. HILL.
A. K. PARPART.
THE APPARATUS COMMITTEE
THE SUPPLY DEPARTMENT COMMITTEE
L. G. EARTH, Chairman.
E. G. BALL.
P. S. GALSOFF.
R. T. KEMPTON.
D. A. MARSLAND.
THE EVENING LECTURE COMMITTEE
B. H. WILLIER, Chairman.
M. H. JACOBS.
CHARLES PACKARD.
H. B. GOODRICH, Chairman.
W. C. ALLEE.
S. C. BROOKS.
VIKTOR HAMBURGER.
CHARLES PACKARD.
THE INSTRUCTION COMMITTEE
II. ACT OF INCORPORATION
No. 3170
COMMONWEALTH OF MASSACHUSETTS
Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T.
Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells,
William G. Farlow, Anna D. Phillips and B. H. Van Vleck have associated themselves
with the intention of forming a Corporation under the name of the Marine Biological
Laboratory, for the purpose of establishing and maintaining a laboratory or station for
scientific study and investigation, and a school for instruction in biology and natural his-
tory, and have complied with the provisions of the statutes of this Commonwealth in such
case made and provided, as appears from the certificate of the President, Treasurer, and
Trustees of said Corporation, duly approved by the Commissioner of Corporations, and
recorded in this office ;
Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu-
setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi-
ner. S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck,
their associates and successors, are legally organized and established as, and are hereby
MARINE BIOLOGICAL LABORATORY
made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB-
ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties,
and restrictions, which by law appertain thereto.
Witness my official signature hereunto subscribed, and the seal of the Commonwealth
of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord
One Thousand Eight Hundred and Eighty-Eight.
[SEAL]
HENRY B. PIERCE,
Secretary of the Connnomvealtli.
III. BY-LAWS OF THE CORPORATION OF THE MARINE
BIOLOGICAL LABORATORY
I. The annual meeting of the members shall be held on the second Tuesday in August,
at the Laboratory, in Woods Hole, Mass., at 11.30 A.M., daylight saving time, in each
year, and at such meeting the members shall choose by ballot a Treasurer and a Clerk to
serve one year, and eight Trustees to serve four years. There shall be thirty-two Trustees
thus chosen divided into four classes, each to serve four years, and in addition there shall
be two groups of Trustees as follows : (a) Trustees ex officio, who shall be the President
of the Corporation, the Director of the Laboratory, the Associate Director, the Treasurer
and the Clerk; (b) Trustees Emeritus, who shall be elected from the Trustees by the Cor-
poration. Any regular Trustee who has attained the age of seventy years shall continue
to serve as Trustee until the next annual meeting of the Corporation, whereupon his office
as regular Trustee shall become vacant and be filled by election by the Corporation and he
shall become eligible for election as Trustee Emeritus for life. The Trustees ex officio
and Emeritus shall have all rights of the Trustees except that Trustees Emeritus shall not
have the right to vote.
The Trustees and officers shall hold their respective offices until their successors are
chosen and have qualified in their stead.
II. Special meetings of the members may be called by the Trustees to be held in Boston
or in Woods Hole at such time and place as may be designated.
III. Inasmuch as the time and place of the Annual Meeting of Members are fixed by
these By-laws, no notice of the Annual Meeting need be given. Notice of any special
meeting of members, however, shall be given by the Clerk by mailing notice of the time
and place and purpose of said meeting, at least fifteen (15) days before such meeting, to
each member at his or her address as shown on the records of the Corporation.
IV. Twenty-five members shall constitute a quorum at any meeting.
V. The Trustees shall have the control and management of the affairs of the Corpora-
tion; they shall present a report of its condition at every annual meeting; they shall elect
one of their number President of the Corporation who shall also be Chairman of the Board
of Trustees; they shall appoint a Director of the Laboratory; and they may choose such
other officers and agents as they may think best ; they may fix the compensation and define
the duties of all the officers and agents ; and may remove them, or any of them, except those
chosen by the members, at any time ; they may fill vacancies occurring in any manner in
their own number or in any of the offices. They shall from time to time elect members
to the Corporation upon such terms and conditions as they may think best.
VI. Meetings of the Trustees shall be called by the President, or by any two Trustees,
and the Secretary shall give notice thereof by written or printed notice sent to each Trustee
by mail, postpaid. Seven Trustees shall constitute a quorum for the transaction of busi-
ness. The Board of Trustees shall have power to choose an Executive Committee from
their own number, and to delegate to such Committee such of their own powers as they
may deem expedient.
REPORT OF THE TREASURER
VII. The accounts of the Treasurer shall be audited annually by a certified public ac-
countant.
VIII. The consent of every Trustee shall be necessary to dissolution of the Marine
Biological Laboratory. In case of dissolution, the property shall be disposed of in such
manner and upon such terms as shall be determined by the affirmative vote of two-thirds
of the Board of Trustees.
IX. These By-laws may be altered at any meeting of the Trustees, provided that the
notice of such meeting shall state that an alteration of the By-laws will be acted upon.
X. Any member in good standing may vote at any meeting, either in person or by
proxy duly executed.
IV. THE REPORT OF THE TREASURER
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY:
Gentlemen:
Herewith is my report as Treasurer of the Marine Biological Laboratory for
the year 1942.
The accounts have been audited by Messrs. Seamans, Stetson and Tuttle,
certified public accountants. A copy of their report is on file at the Laboratory
and is open to inspection by members of the Corporation.
The principal summaries of their report — The Balance Sheet, Statement of
Income and Expense, and Current Surplus Account — are appended hereto as
Exhibits A, B and C.
The following are some general statements and observations based on the
detailed reports :
/. Assets
1. Endowment Assets
At the end of 1942 the total of all the Endowment Assets was $1,071,990.90,
a loss of $7,821.17 from the preceding total, due largely to the loss of $8.801.13
incurred in the sale of one of the New York City real estate holdings on which
the Laboratory had held a mortgage participation. The market value of the
marketable securities increased slightly during the year. Using book values for
the mortgage and real estate participations for which there are no market values,
the total market value of all Endowment Assets was $1,020,282.41, compared with
$999,599.86 at the end of 1941.
2. Plant Assets
The total of Plant Assets (excluding the Gansett and Devil's Lane Tracts) was
$1,357.761.97 after deduction of $608,146.02 accumulated Depreciation Reserve.
This represents a decrease of $18,407.84. Actual additions to Plant Assets during
the year totalled $12,208.30 but this gain was more than offset by depreciation
charges on buildings and equipment amounting to $26,935.14.
During the year $108.00 was expended on the construction of the Library
addition. This left a balance of $2,762.07 remaining from the gifts totalling
$110,400.00 received in 1940 and 1941 from the Rockefeller Foundation for the
addition. This unexpended balance of $2.762.07 was returned to the Rockefeller
Foundation in December in accordance witb the understanding with the donor.
6 A1ARINE BIOLOGICAL LABORATORY
3. Current Assets
Current Assets, including cash, inventories and investments not in the Endow-
ment Funds, amounted to $164,669.02, a decrease of $7,001.09. Current Liabilities
(accounts payable) were $4,996.85 as compared with $6,423.47 so that Current
Surplus was down only $5,574.47 to a total of $159,672.77.
II. Income and Expenditures
Total Income was $146,069.76, a decrease of $16,676.94 from 1941. Total
expenditures including the $26,935.14 added to Depreciation Reserves were $163,-
281.69, a decrease of $8,947.02. The deficit for the year was, therefore, $17,211.93
as compared with the 1941 deficit of $9,482.01.
The decline in income was due to several factors. Income from the General
Endowment and Library Funds was down from $38,879.73 in 1941 to $35,883.81.
Dividends from the General Biological Supply House, Inc., dropped from $17,780.00
to $10,922.00. "Research" net income declined from $8,606.65 to $4,948.42.
"Instruction" resulted in a net loss of $2,063.57 instead of the 1941 net profit
of $176.45.
The Laboratory Administration met the problem created by reduced income by
reducing operating expenses as shown in the detailed appendices. Maintenance
expenses were substantially reduced and the usual deficits in operation of the mess,
dormitory and supply departments (deficits caused only by depreciation and rental
charges) were lessened. The rentals received from the United States Navy for
the Laboratory properties under lease (Mess Hall, Apartment House, etc.) were
also of assistance in reducing the deficit. Such rentals actually paid in 1942
amounted to $10.847.47 and were allocated to the respective accounts. In addition
as of December 31st there were rental accruals clue from the Navy of $1,677.47.
EXHIBIT A
MARINE BIOLOGICAL LABORATORY BALANCE SHEET, DECEMBER 31, 1942
Assets
Endowment Assets and Equities :
Securities and Cash in Hands of Central Hanover Bank and
Trust Company, New York, Trustee $1,062,364.97
Securities and Cash in Minor Funds 9,625.93
$1,071.990.90
Plant Assets :
Land $ 111,425.38
Buildings 1,322,315.51
Equipment 185,313.69
Library 320,069.89
$1,939.124.47
Less Reserve for Depreciation 608,146.02
$1,330,978.45
Cash in Reserve Fund 4,273.51
Cash in Book Fund . 22,510.01
$1,357,761.97
REPORT OF THE TREASURER
Current Assets :
Cash $ 4.965.40
Accounts Receivable 18.537.86
Inventories :
Supply Department $ 31.683.18
Biological Bulletin 12.768.29
$ 44,451.47
Investments :
Devil's Lane Property $ 45,720.27
Gansett Property 100.68
Stock in General Biological Supply House,
Inc 12,700.00
Other Investment Stocks 17,770.00
Retirement Fund 14,137.88
$ 90,428.83
Prepaid Insurance 4,291.72
Items in Suspense 1,994.34
$ 164,669.62
Total Assets $2,594,422.49
Liabilities
Endowment Funds :
Endowment Funds $1,060.069.32
Reserve for Amortization of Bond Premiums.. 2.295.65
$1,062.364.97
Minor Funds 9,625.93
$1,071,990.90
Plant Liabilities and Surplus :
Donations and Gifts $1,172,564.04
Other Investments in Plant from Gifts and Current Funds . 185,197.93
$1,357,761.97
Current Liabilities and Surplus :
Accounts Payable $ 4,996.85
Current Surplus (Exhibit C) 159,672.77
$ 164,669.62
Total Liabilities $2,594,422.49
EXHIBIT B
MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE,
YEAR ENDED DECEMBER 31, 1942
Income :
Total Net
Expense Income Expense Income
General Endowment Fund $ 29,549.85 $ 29,549.85
Library Fund 6,333.96 6,333.96
Instruction $ 7,508.57 5,445.00 $ 2,063.57
Research 5,600.98 10,549.40 4,948.42
8
MARINE BIOLOGICAL LABORATORY
Evening Lectures 8.95
Biological Bulletin and Membership Dues.. 7,575.89
Supply Department 42,306.45
Mess 16,040.39
Dormitories 21,151.71
(Interest and Depreciation charged to above
3 Departments) 24,197.25
Dividends, General Biological Supply House,
Inc
Dividends, Crane Company
Rents :
Bar Neck Property 648.80
Janitor House 24.16
Danchakoff Cottages 270.11
Lecture Hall and Botany Building
Sale of Library Duplicates and Micro Films
Apparatus Rental
Sundry Income
Maintenance of Plant :
Buildings and Grounds 21,419.05
Apparatus Department 5,952.23
Chemical Department 2,789.77
Library Expense 7,883.29
Workmen's Compensation Insurance .... 541.87
Truck Expense 307.04
Bay Shore Property 86.57
Great Cedar Swamp 19.35
General Expenses :
Administration Expense 12,501.53
Endowment Fund Trustee and Safe-
keeping 1,014.45
Bad Debts 355.78
Special Repairs, Supply Dep't Stone Build-
ing 5,811.86
Payment to former Technical Director .... 725.00
Reserve for Depreciation 26,935.14
8,244.44
40,607.40
14,436.11
12,676.29
10,922.00
500.00
4,338.02
360.00
600.00
666.66
89.82
689.63
60.28
8.95
1,699.05
1,604.28
8,475.42
668.55
21,419.05
5,952.23
2,789.77
7,883.29
541.87
307.04
86.57
19.35
12,501.53
1,014.45
355.78
5,811.86
725.00
26,935.14
24,197.25
10,922.00
500.00
3,690.12
335.84
329.89
666.66
89.82
689.63
60.28
Excess of Expense over Income carried to
Current Surplus
$163,281.69 $146.069.76 $100,194.20 $ 82,982.27
$ 17,211.93
$163,281.69
$ 17,211.93
$100,194.20
EXHIBIT C
MARINE BIOLOGICAL LABORATORY, CURRENT SURPLUS ACCOUNT
YEAR ENDED DECEMBER 31, 1942
Balance, January 1, 1942 $165,247.24
Add:
Reserve for Depreciation Charged to Plant Funds $26,935.14
Bad Debts Recovered 36.39
Gain on Gansett Lot Sold 47.83
$ 27,019.36
$192,266.60
REPORT OF THE LIBRARIAN
Deduct :
Excess of Expense over Income for Year as shown in Exhibit B. . $17,211.93
Payments from Current Funds during Year for Plant Assets :
Buildings $ 3,192.99
Equipment 1,580.98
Library 7,449.33
$12,223.30
Less Received for Plant Assets Disposed of 15.00
$12,208.30
Pensions Paid $ 3,460.00
Less Retirement Fund Income 286.40
$ 3,173.60
$ 32,593.83
Balance, December 31, 1942 $159,672.77
Respectfully Submitted,
DONALD M. BRODIE,
Treasurer.
V. REPORT OF THE LIBRARIAN
The Library budget for 1942 was greatly reduced by action of the Executive
Committee. For the years 193-1 — 41 inclusive it was $18,850 per year, with only
slight variations; for 1942 it was $12,200, a decrease of more than $6,000. Since
1940 we have received fewer and fewer European continental journals, until now
practically none come in. Our subscriptions, however, are kept up, and the jour-
nals which cannot be delivered are being stored for the duration. Meanwhile no
payments for these subscriptions have been made. For this reason there was an
unexpended balance at the end of 1940. In 1940 the Library Committee requested
that the balance, amounting to $3,977.18, be placed in a reserve fund from which
to pay for the journals and back sets at such a time as they might be delivered.
This request was granted by the Executive Committee. Early in the year 1941 a
sum of $2,228.32 wras so spent. A similar request in 1941 was not granted, and
the unexpended balance of $2,663.48 for that year reverted to the general fund of
the Laboratory. No request for a reserve fund was made in 1942. The Labora-
tory is now committed to pay for three years of foreign subscriptions, assuming that
the journals can be delivered at some future time. There is now no adequate re-
serve fund from which such payments may be made.
This year the $12,200 appropriated was expended as follows: books, $91.06;
serials, $1,489.28; binding, $1,084.05; express, $174.63; supplies, $471.15; sal-
aries, $7,200; back sets, $1,797.92; sundries, $26.75; and insurance, $45.00; total,
$12,379.84. The sales of duplicates brought in this year $26.06 and the income
from the microfilm service inaugurated in the summer amounted to $63.76, the
expenses for this latter having been charged to "supplies" and "salaries."
From the "Carnegie Fund" $2,239.01 was spent for back sets and journals and
$250.98 for valuable books that we term biological "classics"; in all, 15 completed
back sets, 24 partially completed and 23 "classics."
10 MARINE BIOLOGICAL LABORATORY
The Woods Hole Oceanographic Institution appropriated $800 to the Library
for 1942 and a balance of $154.65 remained from the 1941 budget. An expended
sum of $884.54 has been reported to the Director. A balance of $70.11 was carried
on to the year 1943.
Since practically no current issues of journals have come to us from Europe
since June 1941, it seems best in this report to give the figures for current journals
actually received rather than for the subscriptions and exchange orders due us.
This explains the sharp drop in this item that follows as compared with that for
1941. In 1942 the Library received 637 current publications (1,297 in 1941) : 227
(11 new) in subscriptions to the Marine Biological Laboratory, 18 (1 new) to the
Woods Hole Oceanographic Institution; 209 exchanges, 192 (2 new) with the
"Biological Bulletin" and 17 (0 new) with the Woods Hole Oceanographic Institu-
tion publications; 178 gifts to the former and 5 to the latter. The Marine Biologi-
cal Laboratory acquired 107 books ; 43 by purchase of the Marine Biological Labora-
tory (23 "classics" see above), 15 by purchase of the Woods Hole Oceanographic
Institution; 16 as gifts from the authors, 30 from publishers and 3 miscellaneous.
There were 47 back sets of serial publications completed ; 34 purchased by the
Marine Biological Laboratory (1 5 with "Carnegie Fund") ; 2 by the Woods Hole
Oceanographic Institution ; 1 secured by exchange of the "Biological Bulletin" ; 5
as gifts to the same and 5 by exchange of duplicate material. Partially completed
sets were 164: purchased by the Marine Biological Laboratory, 59 (24 with "Car-
negie Fund") ; by the Woods Hole Oceanographic Institution, 3; by exchange of
the "Biological Bulletin," 1 ; by gift to the same, 44 ; and by exchange of duplicate
material, 57. The reprint additions number 3,097: current of 1941, 436; current
of 1942, 23 ; and of previous dates, 2,638. The present holdings of the Library
are 50,937 bound volumes and 122,723 reprints.
Very few of the current reprints received were catalogued during 1942. From
May until November three members of the staff spent the major part of their work-
ing hours on the "List of serial holdings" to be published as a "supplement" to the
"Biological Bulletin" in the February 1943 issue. The total reprints of date 1941
therefore will be recorded, as well as those of the date 1942, in the 1943 report.
The current reprints separated from those of previous dates were first counted in
the 1937 report and are summarized as follows: 1936-37, 4,602; 1938, 2,453; 1939,
2,246; and 1940, 1,887. The decline in current reprints in 1940 continues in
1941-42. It would seem that the efforts made so far by the Librarian to impress
upon investigators the importance of these current reprints can have had no sus-
tained effect. The best results were obtained by personal interviews of the Library
with individuals and credit must be given to those, and they are considerable in
number, who do conscientiously send their reprints as issued. Perhaps a better
method of keeping the collection to date may be devised when the war conditions
are over.
During the year seven valuable gifts in non-current reprints were received ; a
total of 17,017. Of these 7,759 were new to us and will be filed for use ; 9,258, be-
ing duplicate, will be placed in our duplicate files and any third copies will be for
sale. The Library is indebted to Dr. Rudolf Hober for the generous gift of his
collection of 7,217 reprints in the subjects of physical and physiological chemistry
and physiology; to Dr. H. E. Crampton, for the high figure of 5,102 reprints on
miscellaneous subjects; from Mrs. H. J. Fry and Dr. Robert Chambers, Dr. Fry's
REPORT OF THE DIRECTOR 11
collection in cytology, 2,660 in all; Dr. D. J. Edwards contributed 606 reprints;
Dr. E. J. Herrick, 383; Dr. Libbie H. Hyman, 965; and Dr. B. M. Davis, 84.
Miss Mathilda Koch kindly sent to us several sets of journals and four books from
the Library of her brother, Dr. Waldemar Koch, with the understanding that the
books should be incorporated in the Library, and the journals, which are duplicates
to us, should revert to our use for sale or exchange in case the sale of these is not
consummated within a given period. The addition of 7,759 reprints to the back
files of reprints is the highest number that has ever been added in one year to the
Library's collection and Dr. Hober's gift is the largest single collection that this
Library has ever received.
VI. THE REPORT OF THE DIRECTOR
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY :
Gentlemen:
I beg to submit herewith a report of the fifty-fifth session of the Marine Biologi-
cal Laboratory for the year 1942.
1. Changes in Personnel. At the Trustees' meeting in 1940, Dr. F. R. Lillie
presented his resignation as President of the Corporation and Chairman of the
Executive Committee. He was persuaded, however, to continue his duties until
suitable preparations could be made for naming his successor. The Committee
entrusted with this responsibility agreed that the office of President deals largely
with the external relations of the Laboratory and that search should be made for
some one who would appropriately represent the Laboratory in this field. At the
same time they felt that there should be a Vice President who would represent
Biology. After clue consideration, Lawrason Riggs, Treasurer of the Corporation
since 1924, was nominated for the Presidency, and Dr. E. Newton Harvey was
named to fill the newly created position of Vice President. They were formally
elected to these < ffires at the Trustees' meeting in 1942. At the Corporation meet-
ing, Mr. Donald M. Brodie, formerly manager of Mr. C. R. Crane's New York
office, was elected Treasurer in place of Mr. Riggs, and Dr. Otto Glaser was elected
Clerk of the Corporation in place of Dr. P. B. Armstrong who resigned because of
pressure of war work. The Laboratory is fortunate in securing the services of
these men and confidently gives them its whole-hearted support. Dr. Lillie was
named President Emeritus of the Corporation. Dr. Packard, the Director, was
made Resident Director, and assumed his full time duties at the Laboratory on
October 1, 1942.
2. Financial. At the present time the financial condition of the Laboratory is
satisfactory, even though our income has fallen during the past few years. In
1942 it was about 16 per cent below the average of the preceding eight years, the
decrease being due, in large measure, to a sharp drop in returns from the endow-
ment funds and from dividends, from research fees, and from the courses of in-
struction. It is a matter of gratification that many of our subscribing institutions
have continued their support even though they can send few representatives or
none at all. On the other hand, the income from the Supply Department increased,
and so also did the item of Rentals, a result of the Navy's occupation of the Apart-
ment House, the Mess, and other buildings. At the same time our expenditures
have been reduced. The cost of maintaining the buildings, and of administration,
12 MARINE BIOLOGICAL LABORATORY
has fallen somewhat below the average, but the chief reductions are in the appropri-
ations for the Library and the Apparatus Department. These economies have, in a
sense, been forced upon us. A large proportion of the foreign journals, to which
we are still subscribing, can no longer be delivered to us; our payments for them
have therefore ceased. Then, also, we can buy little new scientific equipment.
Thus far this has not worked any great hardship on the investigators, for with
reduced attendance, the call for apparatus has lessened. What we have on hand
can be adapted to new needs with the aid of the Apparatus Department staff. Un-
der these conditions the Laboratory can continue to operate within its budget.
But these conditions will not long continue. When normal interchange with
Europe is re-established we shall presumably receive the journals now held in
storage for us, and for them we must pay approximately the amount which was
taken from the Library appropriation. So, also, after the war we shall need a sub-
stantial sum for the replacement of old apparatus, and more particularly, for the
purchase of new tools for research to be used in new fields, such as electronics, which
have been so greatly developed within the last few years.
3. Attendance. A comparison of the attendance at the Laboratory in 1941 and
1942 with the average of the preceding five-year period shows how seriously the war
is affecting us. The years 1936-1940 marked the highest attendance in our history.
In 1937 the total registration was 511, and in 1940, 507. The decline began in
1941 when the number of independent investigators fell off noticeably. The other
groups, however, were present in normal numbers. In 1942 the attendance in all
groups declined sharply, the change being most marked among the younger mem-
bers. Only about one-third of the usual number of assistants was present, and
only one-fourth of the beginning investigators. In the classes, attendance dropped
to about two-thirds of the average except in Physiology where the falling off was
greater. Many of the investigators taught at their colleges throughout the sum-
mer, and will continue to do so for the duration. Others are engaged on wartime
problems which they are carrying on at their own institutions. In many cases this
research is in their chosen field, so their time is by no means lost. Indeed in some
instances it has already opened up new fields for future exploration. But the armed
services have absorbed a large proportion of the younger generation who normally
would take our courses or begin research. For some time to come, few young and
vigorous minds will be added to our list of investigators. All the more it is en-
cumbent upon those who remain to continue and to extend their peacetime investi-
gations.
RECORD OF ATTENDANCE, 1936-1942
1936 1937 1938 1939 1940 Ave. 1941 1942
Independent Investigators 226 256 246 213 253 239 228 16.0
Assistants 57 61 81 79 71 70 50 25
Beginning Investigators 76 74 53 60 62 65 59 16
Students 138 133 132 133 128 133 131 74
Corrected Totals 473 511 496 471 507 489 461 273
4. Losses by Death. In the death of Dr. Calkins the Laboratory loses a de-
voted friend. His important services as Clerk of the Corporation, Secretary of the
Board of Trustees, as an active member of many committees, and as head of the
Protozoology course, will long be remembered.
REPORT OF THE DIRECTOR 13
During the year also occurred the death of one of our Life Members, Dr. A. Law-
rence Lowell, who served as Clerk of the Corporation from 1890 to 1894.
5. The Stone Building. During the fall and winter the Stone Building has been
completely renovated. The decision to do this was made when the Executive Com-
mittee, after a tour of inspection, realized how serious the condition was. Only a
part of the basement could be used ; the stairs were no longer safe ; the first and
second floors were not strong enough to permit the storage of heavy tanks ; the
shingles and trim were in bad shape. These deficiencies have been corrected. The
entire basement is now available for storage, there is a new concrete floor, a new
heating system, and adequate plumbing and lighting. To provide more head room,
the ceiling was raised 18 inches. Many steel columns, both in the basement and in
the first floor, support the great carrying beams which are still sound. The front of
the first floor is now divided into offices and laboratories. The business of the Sup-
ply Department can therefore be carried on in the Stone building, leaving the wooden
building to be used primarily for the preparation of material. These changes were
planned and carried out by Mr. Larkin and Mr. Hemenway, the latter bearing the
larger share of the work. Both the inside and the outside of the building are now
in excellent condition.
In summary, the Laboratory during these disturbing times is maintaining its
usual services, and by rigid economies, is balancing its budget. This quiescent pe-
riod will not long continue ; we must prepare for an expansion of our research
facilities soon after the war ends.
6. Election of Trustees. At the meeting of the Corporation held August 11,
1942, the following Trustees were elected Trustees Emeritus :
A. P. Mathews, University of Cincinnati
S. O. Mast, The Johns Hopkins University
The new Trustees elected at that meeting are :
Eugene F. DuBois, Class of 1944
Eric G. Ball, Class of 1944
7. There are appended as parts of this report :
1. The Staff.
2. Investigators and Students.
3. Tabular View of Attendance, 1938-1942.
4. Subscribing and Co-operating Institutions.
5. Evening Lectures.
6. Shorter Scientific Papers.
7. Members of the Corporation.
Respectfully submitted,
CHARLES PACKARD,
Director
1. THE STAFF, 1942
CHARLES PACKARD, Director, Marine Biological Laboratory, Woods Hole, Massachusetts.
SENIOR STAFF OF INVESTIGATION
GARY N. CALKINS, Professor Emeritus of Protozoology, in residence, Columbia Univer-
sity.
14 MARINE BIOLOGICAL LABORATORY
E. G. CONKLIN, Professor of Zoology, Emeritus, Princeton University.
CASWELL GRAVE, Professor of Zoology, Emeritus, Washington University.
FRANK R. LILLIE, Professor of Embryology, Emeritus, The University of Chicago.
RALPH S. LILLIE, Professor of General Physiology, The University of Chicago.
C. E. McCLUNG, Professor of Zoology, Emeritus, University of Pennsylvania.
S. O. MAST, Professor of Zoology, Johns Hopkins University.
A. P. MATHEWS, Professor Emeritus, Biochemistry, University of Cincinnati.
T. H. MORGAN, Director of the Biological Laboratory, California Institute of Technology.
G. H. PARKER, Professor of Zoology, Emeritus, Harvard University.
ZOOLOGY
I. CONSULTANTS
T. H. BISSONNETTE, Professor of Biology, Trinity College.
L. L. WOODRUFF, Professor of Protozoology, Yale University.
II. INSTRUCTORS
A. J. WATERMAN, Associate Professor of Biology, Williams College, in charge of course.
JOHN B. BUCK, Assistant Professor of Zoology, University of Rochester.
M. D. BURKENROAD, Assistant Curator, Bingham Oceanographic Foundation, Yale Uni-
versity.
W. G. HEWATT, Professor of Biology, Texas Christian University.
W. E. MARTIN, Associate Professor of Zoology. DePauw University.
N. T. MATTOX, Assistant Professor of Zoology, Miami University.
R. W. WILHELMI, Instructor in Zoology, University of Missouri.
III. LABORATORY ASSISTANT
RUTH MERWIN, University of Chicago.
EMBRYOLOGY
I. CONSULTANTS
L. G. BARTH, Assistant Professor of Zoology, Columbia University.
H. B. GOODRICH, Professor of Biology, Wesleyan University.
II. INSTRUCTORS
VIKTOR HAMBURGER, Professor of Zoology, Washington University, in charge of course.
DONALD P. COSTELLO, Assistant Professor of Zoology, University of North Carolina (ab-
sent in 1942).
CHARLES B. METZ, Teaching Fellow, California Institute of Technology.
OLIN RULON, Assistant Professor of Biology, Wayne University.
RAY L. WATTERSON, Instructor in Embryology, Dartmouth College.
PHYSIOLOGY
I. CONSULTANTS
WILLIAM R. AMBERSON, Professor of Physiology, University of Maryland, School of
Medicine.
HAROLD C. BRADLEY, Professor of Physiological Chemistry, University of Wisconsin.
WALTER E. GARREY, Professor of Physiology, Vanderbilt University Medical School.
MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania.
REPORT OF THE DIRECTOR 15
II. INSTRUCTORS
RUDOLF T. KEMPTON, Professor of Zoology, Vassar College, in charge of course.
KENNETH C. FISHER, Assistant Professor of Experimental Biology, University of To-
ronto.
ARTHUR C. GIESE, Associate Professor of Biology, Stanford University.
F. J. M. SICHEL, Assistant Professor of Physiology, University of Vermont, College of
Medicine.
BOTANY
I. CONSULTANTS
S. C. BROOKS, Professor of Zoology, University of California.
D. R. GODDARD, Assistant Professor of Botany, University of Rochester.
II. INSTRUCTORS
WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan, in charge of
course.
HANNAH CROASDALE, Technical Assistant, Dartmouth College.
EXPERIMENTAL RADIOLOGY
G. FAILLA, Memorial Hospital, New York City.
L. ROBINSON HYDE, Phillips Exeter Academy, Exeter, N. H.
LIBRARY
PRISCILLA B. MONTGOMERY (MRS. THOMAS H. MONTGOMERY, JR.), Librarian
DEBORAH LAWRENCE MARY A. ROHAN S. MABELL THOMBS
APPARATUS DEPARTMENT
E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager
J. D. GRAHAM R. S. LILJESTRAND
CHEMICAL DEPARTMENT
KENNETH C. BALLARD, Lawrence High School, Falmouth, Mass., Manager
SUPPLY DEPARTMENT
JAMES MC!NNIS, Manager
RUTH CROWELL GRACE HARMAN
M. B. GRAY W. E. KAHLER G. LEHY
A. M. HILTON A. W. LEATHERS F. N. WHITMAN
GENERAL OFFICE
F. M. MACNAUGHT, Business Manager
POLLY L. CROWELL GLADE F. ALLEN
GENERAL MAINTENANCE
T. E. LARKIN, Superintendent
F. A. CANNON T. E. TAWELL
W. C. HEMENWAY R. F. TRAVIS
R. W. KAHLER J. WYNNE
THE GEORGE M. GRAY MUSEUM
GEORGE M. GRAY, Curator Emeritus
16 MARINE BIOLOGICAL LABORATORY
2. INVESTIGATORS AND STUDENTS
Independent Investigators, 1942
ADDISON, WILLIAM H. F., Professor of Normal Histology and Embryology, University of
Pennsylvania, School of Medicine.
ANDERSON, THOMAS F., RCA Fellow, National Research Council.
ANDREW, WARREN, Assistant-Professor of Histology and Embryology, Baylor University, Col-
lege of Medicine.
BAKER, HORACE B., Professor of Zoology, University of Pennsylvania.
BALL, ERIC G., Associate Professor, Department of Biological Chemistry, Harvard Medical
School.
BALL, ERNEST, National Research Fellow in Botany, Yale University.
BARTH, L. G., Assistant Professor of Zoology, Columbia University.
BARTLETT, JAMES H., JR., Associate Professor of Theoretical Physics, University of Illinois.
BERGER, CHARLES A., Professor of Cytology and Genetics, Fordham University.
BISSONNETTE, T. H., Professor of Biology, Trinity College.
BLUM, JOHN L., Instructor in Biology, Canisius College.
BODIAN, DAVID, Associate in Epidemiology, Johns Hopkins University.
BOTSFORD, E. FRANCES, Associate Professor of Zoology, Connecticut College.
BROOKS, MATILDA M., Research Associate, University of California.
BROOKS, SUMNER C., Professor of Zoology, University of California.
BUCK, JOHN B., Assistant Professor of Zoology, University of Rochester.
BUDINGTON, R. A., Professor of Zoology, Emeritus, Oberlin College.
BURKENROAD, MARTIN D., Assistant Curator, Peabody Museum, Yale University.
CANNAN, R. KEITH, Professor, New York University College of Medicine.
CHAMBERS, ROBERT, Research Professor of Biology, Washington Square College, New York
University
CHENEY, RALPH H., Professor of Biology, Long Island University.
CHILD, RUTH C., Assistant Professor, Wellesley College.
CLARK, ELEANOR L., Department of Anatomy, University of Pennsylvania.
CLARK, ELIOT R., Professor and Director of Department of Anatomy, University of Pennsyl-
vania, School of Medicine.
CLOWES, G. H. A., Director of Research, Eli Lilly and Company.
CON KLIN, EDWIN G., Professor of Biology, Emeritus, Princeton University.
COPELAND, MANTON, Professor of Biology, Bowdoin College.
CROASDALE, HANNAH T., Technical Assistant, Dartmouth College.
DELBRUCK, MAX, Instructor in Physics, Vanderbilt University.
DREYER, NICHOLAS B., Associate Professor of Pharmacology, Long Island College of Medicine.
EAKIN, RICHARD M., Assistant Professor of Zoology, University of California.
ELIZABETH, SISTER MIRIAM, Associate Professor of Biology, Chestnut Hill College.
EVANS, TITUS C., Research Assistant Professor of Radiology, State University of Iowa.
FAILLA, G., Physicist, Memorial Hospital.
FISHER, KENNETH C., Assistant Professor of Physiological Zoology, University of Toronto.
FREY, DAVID G., Junior Aquatic Biologist, U. S. Fish and Wildlife Service.
FRISCH, JOHN A., Professor of Biology, Head of Biology Department, Canisius College.
GABRIEL, MORDECAI L., Lecturer in Zoology, Columbia University.
GALTSOFF, PAUL S., Biologist in Charge Shellfish Investigation, U. S. Fish and Wildlife
Service.
GARREY, W. E., Professor of Physiology, Vanderbilt University School of Medicine.
GIESE, ARTHUR C., Associate Professor of Biology, Stanford University.
GLASER, OTTO C., Professor of Biology, Amherst College.
GRAND, C. G., Research Associate, Washington Square College, New York University.
GRAVE, CAS WELL, Professor of Zoology, Emeritus, Washington University.
GUREWICH, VLADIMIR, Clinical Assistant and Attending Physician, Cornell Division of the
Bellevue Hospital.
HAMBURGER, VIKTOR, Professor of Zoology, Washington University.
REPORT OF THE DIRECTOR 17
HARTMAN, FRANK A., Professor and Chairman Department of Physiology, Ohio State Uni-
versity.
HARVEY, ETHEL B., Research Investigator, Princeton University.
HARVEY, E. NEWTON, Professor of Physiology, Princeton University.
HAUGAARD, G., Research Assistant, Harvard University.
HAYWOOD, CHARLOTTE, Professor of Physiology, Mount Holyoke College.
HEILBRUNN, L. V., Associate Professor of Zoology, University of Pennsylvania.
HENRY, RICHARD J., Medical Student, School of Medicine, University of Pennsylvania.
HEWATT, WILLIS G., Professor of Biology, Texas Christian University.
HILL, SAMUEL E., Professor of Biology, Russell Sage College.
HOPKINS, HOYT S., Associate Professor of Physiology, New York University College of Den-
tistry.
HOWE, H. E., Editor, Industrial and Engineering Chemistry.
HYMAN, CHESTER, Research Assistant, New York University.
JACOBS, M. H., Professor of General Physiology, University of Pennsylvania Medical School.
JOHLIN, J. M., Associate Professor, Vanderbilt University School of Medicine.
KEMPTON, RUDOLF T., Professor of Zoology, Vassar College.
KNOWLTON, FRANK P., Professor of Physiology, Syracuse University, College of Medicine.
KOPAC, M. J., Visiting Assistant Professor of Biology, New York University.
KRAHL, M. E., Research Chemist, Eli Lilly and Company.
LILLIE, FRANK R., Professor of Embryology, Emeritus, The University of Chicago.
LILLIE, RALPH S., Professor of Physiology, Emeritus, The University of Chicago.
LITTLE, ELBERT P., Instructor in Science, Phillips Exeter Academy.
LOWENSTEIN, B. E., Research Associate, New York University, Washington Square College.
LURIA, SALVADOR E., Research Assistant in Surgical Bacteriology, Columbia University.
McBRiDE, ARTHUR F., Curator, Marine Studios Inc.
McCLUNG, C. E., Professor of Zoology, Emeritus, University of Pennsylvania.
MARSLAND, DOUGLAS A., Assistant Professor of Biology, Washington Square College, New
York University.
MARTIN, WALTER E., Associate Professor of Zoology, DePauw University.
MAST, S. O., Professor of Zoology, Johns Hopkins University.
MATHEWS, A. P., Professor of Biochemistry, Emeritus, University of Cincinnati.
MATTOX, N. T., Assistant Professor of Zoology, Miami University.
MAYOR, JAMES W., Professor of Biology, Union College.
MEMHARD, ALLEN R., Crescent Rd., Riverside, Connecticut.
MENKIN, VALY, Assistant Professor of Pathology, Harvard Medical School.
METZ, CHARLES W., Head, Department of Zoology, University of Pennsylvania.
MOLTER, JOHN A., Graduate Student, University of Pennsylvania.
MOOG, FLORENCE, Graduate Student, Columbia University.
MORGAN, T. H., Professor of Biology, California Institute of Technology.
NABRIT, S. MILTON, Professor of Biology, Atlanta University.
NACHMANSOHN, DAVID, Research Associate, Columbia University.
O'BRIEN, JOHN A., Instructor in Biology, Catholic University of America.
OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research.
PACKARD, CHARLES, Director, Marine Biological Laboratory.
PIERSON, BERNICE F., Instructor in Biology, National Park College.
PLOUGH, HAROLD H., Professor of Biology, Amherst College.
POLLISTER, ARTHUR W., Associate Professor of Zoology, Columbia University.
POMERAT, GERARD R., Instructor in Biology, Harvard University.
RICHARDS, A. GLENN, JR., Instructor in Zoology, University of Pennsylvania.
Ris, HANS, Zoology Department, Columbia University.
RUGH, ROBERTS, Associate Professor, Washington Square College, New York University.
RULON, OLIN, Assistant Professor, Wayne University.
RUNYON, ERNEST H., Associate Professor of Botany, Agnes Scott College.
SCHALLEK, WILLIAM B., Biological Laboratories, Harvard University.
SCHAEFFER, A. A., Professor and Chairman of the Department of Biology, Temple University.
18 MARINE BIOLOGICAL LABORATORY
SCOTT, ALLAN C, Assistant Professor of Biology, Union College.
SCOTT, SISTER FLORENCE M., Professor of Zoology, Seton Hill College.
SHANES, ABRAHAM M., Instructor in Physiology, New York University, College of Dentistry.
SHAW, MYRTLE, Senior Bacteriologist, New York State Department of Health.
SHELDEN, FREDERICK F., Instructor in Physiology, Ohio State University.
SICHEL, ELSA KEIL, Head of the Science Department, Vermont State Normal School.
SICHEL, F. J. M., Assistant Professor of Physiology, University of Vermont, College of Medi-
cine.
SIMPSON, JENNIE L. S., Assistant Professor of Botany, Hunter College.
SLIFER, ELEANOR H., Assistant Professor, Department of Zoology, State University of Iowa.
SMELSER, GEORGE K., Assistant Professor of Anatomy, Columbia University College of Physi-
cians and Surgeons.
SPRINGER, STEWART, Marine Studios, Inc.
STEINBACH, H. B., Associate Professor of Zoology, Washington University.
STEWART, DOROTHY R., Associate Professor of Biology, Skidmore College.
STOREY, ALMA G., Professor Emeritus, Mount Holyoke College.
STUNKARD, HORACE W., Professor of Biology, New York University.
TAYLOR, WILLIAM R., Professor of Botany, University of Michigan.
TsWiNKEL, Lois E., Assistant Professor of Zoology, Smith College.
THIVY, FRANCESCA, Graduate Student, University of Michigan.
TRINKAUS, J. PHILIP, Graduate Student, Johns Hopkins University.
TURNER, ABBY H., Professor of Physiology, Emeritus, Mount Holyoke College.
VON SALLMANN, LUDWIG J., Assistant Professor in Ophthalmology, College of Physicians and
Surgeons, Columbia University.
WATERMAN, ALLYN J., Associate Professor of Biology, Williams College.
WENRICH, D. H., Professor of Zoology, University of Pennsylvania.
WENSTRUP, EDWARD J., Head, Department of Biology, St. Vincent College.
WHITING, P. W., Associate Professor of Zoology, University of Pennsylvania.
WIERCINSKI, FLOYD J., Research Assistant, University of Pennsylvania.
WILBUR, KARL M., Instructor, Ohio State University.
WILHELMI, RAYMOND W., Instructor in Zoology, University of Missouri.
WILLIER, B. H., Professor of Zoology, The Johns Hopkins University.
WOLF, E. ALFRED, Associate Professor of Biology, University of Pittsburgh.
WOODRUFF, LORANDE L., Professor of Protozoology and Director of the Osborn Zoological
Laboratory, Yale University.
WRINCH, DOROTHY, Visiting Professor, Smith, Amherst and Mt. Holyoke Colleges.
ZWEIFACH, BENJAMIN W., Research Associate in Biology, New York University.
Beginning Investigators
BRUMMER, DONALD L., Student, New York University, College of Medicine.
CLARK, ARNOLD M., Graduate Student, University of Pennsylvania.
COLE, EDITH, Undergraduate Assistant, Pennsylvania College for Women.
DANIEL, SISTER PAUL, Instructor, Chestnut Hill College.
FERGUSON, FREDERICK P., Teaching Assistant, University of Minnesota.
GROSCH, DANIEL S., Assistant Instructor, University of Pennsylvania.
HINCHEY, M. CATHERINE, Instructor in Biology, Temple University.
JAEGER, LUCENA, Graduate Student, Columbia University.
KELTCH, ANNA K., Research Chemist, Eli Lilly and Co.
LEFEVRE, PAUL G., Research Assistant, University of Pennsylvania.
METZ, CHARLES B., Teaching Fellow, California Institute of Technology.
NELSON, LEONARD, Student, University of Pennsylvania.
SOUTHWICK, MILDRED D., Instructor of Plant Science, Vassar College.
TAYLOR, HARRIETT E., Graduate Assistant, Columbia University.
WATTERSON, RAY L., Instructor. Dartmouth College.
WILSON, WALTER L., Graduate Student, University of Pennsylvania.
REPORT OF THE DIRECTOR 19
Research Assistants
ATKINSON, LENETTE R., Research Assistant, Amherst College.
BARBER, AVA J., Senior Student, University of California.
BOND, CHRISTIANA, Secretary, University of Maryland Medical School.
BROWNELL, KATHARINE A., Research Associate, Ohio State University.
BUTLER, MARY K., Research Assistant, University of Pennsylvania.
COOK, ELIZABETH J., Research Assistant, Harvard University.
DYTCHE, MARYON M., Graduate Assistant, University of Pittsburgh.
EHRENFELD, KLARA, Research Assistant, Amherst College.
GARZOLI, RAY F., Graduate Student, University of California.
HEIDENTHAL, GERTRUDE, Research Assistant, University of Pennsylvania.
HOHWIELER, HAROLD J., Graduate Assistant, Washington University.
JACOBS, JOYE E., Research Assistant, University of Maryland Medical School.
KIBRICK, ANDRE C, Teaching Assistant, New York University Medical College.
KIELICH, E. RANDOLPH, Graduate Assistant, Canisius College.
KRUGEI.IS, EDITH J., Research Assistant, Columbia University.
LONG, M. JEANNE, Research Assistant, New York University.
MACHADO, ANGELO L., Research Fellow, Yale University Medical School.
MERRITT, FRANCES A., Laboratory Assistant, Eli Lilly & Co.
PHILLIPS, CLYDE, Assistant in Anatomy, Morehouse College.
SMITH, DOUGLAS F., Research Assistant, Ohio State University.
SPIEGELMAN, S., Research Assistant, Washington University.
STEVENS, HAZEL A., Laboratory Assistant, Eli Lilly and Co.
STEVENS, KATHARINE, Student, Vassar College.
WOODWARD, ARTHUR A., JR., Research Assistant, Wesleyan University.
WURTZ, CHARLES B., Graduate Student Assistant, University of Pittsburgh.
Library Readers, 1942
AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland Medical School.
BECK, L. V., Instructor in Physiology, Hahnemann Medical College.
BELDA, WALTER H., Assistant Professor, Fordham University.
BLOCK, ROBERT, Research Assistant, Yale University.
CASSIDY, HAROLD G., Yale University.
CLARK, HELEN, Instructor in Zoology, Hunter College of the City of New York.
DIAMOND, Louis K., Associate in Pediatrics, Harvard Medical School.
DIAMOND, MOSES, Associate Professor, Columbia University Dental School.
EVERETT, GUY M., Weaver Research Fellow, University of Maryland Medical School.
FOWLER, COLEEN, Johns Hopkins University.
GATES, R. R., Professor, University of London.
HUTCHINGS, Lois M., Teacher of Biology, Weequahic High School.
JONES, ARTHUR W., Research Fellow in Zoology, University of Virginia.
KREEZER, GEORGE L., Assistant Professor of Psychology, Cornell University.
LAVIN, GEORGE, Rockefeller Institute for Medical Research.
LEVINE, PHILIP, Bacteriologist and Serologist, Beth Israel Hospital.
LOEWI, OTTO, Research Professor, New York University College of Medicine.
LUDWIG, FRANCIS W., Instructor, Villanova College.
MEYERHOF, N. OTTO, Research Professor of Biochemistry, University of Pennsylvania.
MITCHELL, PHILIP H., Professor of Biology, Brown University.
NEWELL, JAMES W., Student, Cornell University Medical College.
OSTER, ROBERT H., Assistant Professor of Physiology, University of Maryland Medical School.
RENSHAW, BIRDSEY, Assistant Professor, Oberlin College.
ROBERTS, EDITH, Chairman, Department of Botany, Vassar College.
SEVAG, M. G., Assistant Professor of Biochemistry, University of Pennsylvania School of
Medicine.
SHAPIRO, HERBERT, Instructor in Physiology, Hahnemann Medical College.
SHWARTZMAN, GREGORY, Head of Department of Bacteriology, The Mount Sinai Hospital.
STILES, KARL A., Professor of Biology, Coe College.
20 MARINE BIOLOGICAL LABORATORY
Students, 1942
BOTANY
ARROWSMITH, HAROLD N.. JR., Student, Johns Hopkins University.
BEHNKE, JANE, Student, Wellesley College.
BOOTH, MARY L., Student, Smith College.
HITCHCOCK, MARGARET V., Goucher College.
KINGSLEY, EUNICE L., Assistant Prof, of Botany, Kansas State College.
PAULL, JOHN J., Student, Washington and Jefferson College.
RICHARDSON, EDWARD A., Graduate Assistant, Rutgers University.
YOUNG, MARGARET E., Assistant in Botany, Wellesley College.
EMBRYOLOGY
BEARDSLEY, MARGARET, Smith College.
Boss, MARY B., Goucher College.
BUGGS, CHARLES W., Prof, of Biology and Head, Division of the Sciences, Dillard University.
CARPENTER, ELIZABETH, Graduate Assistant, Mount Holyoke College.
CHURCHILL, WARREN S., Assistant in Zoology, University of Illinois.
COLE, EDITH, Undergraduate Assistant, Pennsylvania College for Women.
DODD, SAMUEL G., Wesleyan University.
DUNN, BARBARA, Graduate Assistant, Wellesley College.
ELIAS, CATHERINE, Volunteer Laboratory Assistant, Connecticut College.
FOSTER, JAMES J., Graduate Assistant, Amherst College.
GAJDUSEK, D. CARLETON, Student, University of Rochester.
GEISLER, SISTER FRANCIS S., S.S.J., Student, Catholic University.
LITTRELL, JUNE L., Assistant, University of Illinois.
MEMHARD, ALLEN R., Crescent Road, Riverside, Conn.
NEWFANG, DOROTHY, Mount Holyoke College.
NICKERSON, MARK, Graduate Assistant, Johns Hopkins University.
PHILBRICK, MADELINE G., Russell Sage College.
POINDEXTER, JOAN, Smith College.
PRODELL, JOHN H., Brothers College of Drew University.
REYER, RANDALL W., Cornell University.
SEITNER, MARGARET M., Hunter College of the City of New York.
SENYARD, JUANITA, Graduate Assistant, Mount Holyoke College.
SHEA, SAMUEL E., JR., Student Laboratory Instructor, Canisius College.
WOOD, MARCIA, Student, Russell Sage College.
PHYSIOLOGY
CHRISTIANSEN, GERTRUDE M., Assistant, Wellesley College.
HARDENBERGH, ESTHER, Student, Mount Holyoke College.
LARSON, VIRGINIA P., Assistant in Physiology, Vassar College.
Low, EVA M., Student, Radcliffe College.
OSTERMAN, GEORGE B., Instructor, Washington and Jefferson College.
POKER, NATHAN, Brooklyn College.
ZOOLOGY
AVILA, ENRIQUE, Compania Administradora del Guano, Lima, Peru.
BENSON, JOHN A., Undergraduate Assistant, Wesleyan University.
BREARLEY, MARGERY, Graduate Student, Mount Holyoke College.
CHRONIAK, WALTER, Massachusetts State College.
COLE, ELSIE L., Heidelberg College.
COLE, M. ETHEL, Teacher, Frick Educational Commission.
COLLARD, LAVERNE E., Oberlin College.
COSBY, EVELYN L., Laboratory Instructor in Botany, University of Richmond.
REPORT OF THE DIRECTOR 21
CREGAR, MARY, Wilson College.
DAUGHADAY, ELEANOR F., Vassar College.
DINTIMAN, SARA MAE, Rutgers University.
DONALDSON, SARA L., Graduate Assistant, Syracuse University.
DOOCHIN, HERMAN D., Student, University of Miami.
FOGG, N. W., Student, American International College.
FOSTER, JAMES J., Graduate Assistant, Amherst College.
FRANKLIN, REV. ROGER G., Prof, of Biology, St. Joseph's Seminary.
HAAS, ELIZABETH, Bennington College.
HUFFORD, VIRGINIA, Oberlin College.
HYDE, JANE E., Student, Radcliffe College.
JOHNSON, VIENO T., 44 Francis Ave., Cambridge, Mass.
KEISTER, MARGARET L., Instructor, Wheaton College.
LESAGE, MAURICE C., Teacher, Society of Divine Word.
LORENTZ, JOHN J., Graduate Student, Fordham University.
MANNY, ELLA T., Sarah Lawrence College.
NEWCOMER, STANLEY, Assistant, Cornell University.
O'RouRK, ANN E., Duke University.
PETERSON, HAROLD L., Student Assistant, Drew University.
PHILBRICK, MADELINE G., Student, Russell Sage College.
RAYNER, HARRIET A., Massachusetts State College.
SAUNDERS, JOHN W., Graduate Assistant, Johns Hopkins University.
SCHMEISSER, ELIZABETH F., Student, Sweet Briar College.
TAFT, EDITH D., Wheaton College.
WATERMAN, GEORGE E., Professor of Biology, Assumption College.
WECKSTEIN, ABRAHAM M., Instructor of Biology, New York University.
WHITE, MARCIA R., Student, Cornell University.
WOOD, MARCIA, Student, Russell Sage College.
3. TABULAR VIEW OF ATTENDANCE
193S 1939 1940 1941 1942
INVESTIGATORS— Total 380 352 386 337 201
Independent 246 213 253 197 132
Under instruction 53 60 62 59 16
Research assistants 81 79 71 50 25
Library readers 31 28
STUDENTS— Total 132 133 128 131 74
Zoology 54 55 55 36
Protozoology (not given after 1940 ) .' 10 12 7
Embryology 34 36 34 37 24
Physiology 22 21 22 24 6
Botany 12 9 10 15 8
TOTAL ATTENDANCE 512 485 514 468 275
Less persons registered as both students and investi-
gators 16 14 7 2
496 471 507 461 273
INSTITUTIONS REPRESENTED— Total 151 162 148 144 126
By investigators 125 132 112 102 83
By students 67 79 72 43
SCHOOLS AND ACADEMIES REPRESENTED
By investigators 4 2 1 5 2
By students 1 2 2 2 0
FOREIGN INSTITUTIONS REPRESENTED
By investigators 14 8 2 3 0
Bv students . 31110
22
MARINE BIOLOGICAL LABORATORY
4. SUBSCRIBING AND CO-OPERATING INSTITUTIONS
1942
Amherst College
Atlanta University
Beth Israel Hospital
Biological Institute, Philadelphia, Pennsyl-
vania
Bowdoin College
Brooklyn College
Brown University
Bryn Mawr College
Canisius College
College of Physicians and Surgeons
Columbia University
Cornell University
Cornell University Medical College
Drew University
Duke University
Fordham University
Frick Educational Commission
Goucher College
Harvard University
Harvard University Medical School
Heidelburg College
Hunter College
Industrial and Engineering Chemistry, of the
American Chemical Society
John and Mary Markle Foundation
Johns Hopkins University
Julius Rosenwald Fund
Eli Lilly and Co.
Long Island University
Marine Studios, Inc.
Massachusetts State College
Morehouse College
Mount Sinai Hospital, New York City
National Research Council
New York State Department of Health
New York University
New York University College of Medicine
New York University Washington Square
College
Oberlin College
Ohio State University
Pennsylvania College for Women
Princeton University
Radcliffe College
Rockefeller Institute for Medical Research
Russell Sage College
Rutgers University
St. Joseph's Seminary, Dunwoodie, New York
Smith College
State University of Iowa
Sweet Briar College
Syracuse University
Tufts College
Union College
University of Cincinnati
University of Illinois
University of Maryland Medical School
University of Missouri
L^niversity of Pennsylvania
University of Pennsylvania School of Medicine
University of Pittsburgh
University of Rochester
Vanderbilt University
Vanderbilt University Medical School
Vassar College
Villanova College
Washington University
Wellesley College
Wesleyan University
Wheaton College
Woods Hole Oceanographic Institution
Yale University
5. EVENING LECTURES, 1942
Friday, June 26
DR. MICHAEL HEIDELBERGER ''Biological Aspects of Immunity and Com-
plement Action."
Friday, July 3
DR. DONALD R. GRIFFIN "Echo Sounding by Flying Bats."
Friday, July 10
DR. R. RUGGLES GATES "The Nucleolus and Phylogeny."
Friday, July 17
DR. E. NEWTON HARVEY "Animal Luminescence."
Friday, July 24
MR. PER HOST "Norway Fights On."
Friday, July 31
REPORT OF THE DIRECTOR
DR. DAVID NACHMANSOHN ''On the Mechanism of Transmission of
Nerve Impulses."
Friday, August 7
PROF. SELMAN A. WAKSMAN "Science in Soviet Russia on the Eve of the
World War."
Friday, August 14
DR. A. GLENN RICHARDS, JR "Electron Microscope Studies of Insect
Structures and Tissues."
Friday, August 21
DR. ROBERT F. GRIGGS "Timber Lines as Indices of Climatic
Change."
Thursday, August 27
DR. OSCAR W. RICHARDS "The Precision of Sectioning with a Micro-
tome."
Friday, August 28
DR. C. W. METZ "Evolutionary Chromosome Changes in Sci-
ara as Shown by the Giant Salivary
Gland Chromosomes."
6. SHORTER SCIENTIFIC PAPERS, 1942
Tuesday, July 21
DR. K. C. FISHER AND
GRACE W. SCOTT "The physiological basis of temperature 'se-
lection' by fish."
DR. J. R. STERN AND
K. C. FISHER "The action of narcotics on oxygen con-
sumption of resting and caffeinized frog
muscle."
DR. A. C. GIESE AND
E. L. TATUM "Effects of vitamins of the B-complex on
respiration of Neurospora mutants."
Tuesday, August 4
MR. SOL SPIEGELMAN "Differential effects on the mass and time
of appearance of regenerants in Tubu-
laria."
Miss FLORENCE MOOG "Some effects of temperature in the regen-
eration of Tubularia."
DR. MORDECAI GABRIEL "The effect of temperature on vertebral vari-
ations in Fundulus heteroclitus."
Tuesday, August 18
DR. DOROTHY WRINCH "The structure of biologically active mem-
branes."
DR. DOUGLAS MARSLAND "The contractile mechanism in unicellular
melanophores."
DR. E. H. RUNYON "The aggregation of separate cells of Dicty-
ostelium to form a multicellular body."
Tuesday, August 25
DR. G. M. EVERETT "Vitamin B, deficiency in the cat." Motion
pictures in color.
DR. T. H. BISSONNETTE "Experimental modification of molts, and
color-changes by controlled lighting of
the Bonaparte weasel."
24 MARINE BIOLOGICAL LABORATORY
7. MEMBERS OF THE CORPORATION, 1942
1. LIFE MEMBERS
ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France.
ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Maryland.
BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York.
BILLINGS, MR. R. C., 66 Franklin Street, Boston, Massachusetts.
CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania.
COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin.
CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New Jersey.
COWDRY, DR. E. V., Washington University, St. Louis, Missouri.
EVANS, MRS. GLENDOWER, 12 Otis Place, Boston, Massachusetts.
FOOT, Miss KATHERINE, Care of Morgan Harjes Cie, Paris, France.
GARDINER, MRS. E. G., Woods Hole, Massachusetts.
JACKSON, MR. CHAS. C., 24 Congress Street, Boston, Massachusetts.
JACKSON, Miss M. C., 88 Marlboro Street, Boston, Massachusetts.
KING, MR. CHAS. A.
KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York.
LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland.
LOWELL, MR. A. L., 17 Quincy Street, Cambridge, Massachusetts.
MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts.
MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Missouri.
MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pa.
MORGAN, MR. J. PIERPONT, JR., Wall and Broad Streets. New York City, New
York.
MORGAN, MRS. T. H., Pasadena, California.
MORGAN, PROF. T. H., Director of Biological Laboratory, California Institute of
Technology, Pasadena, California.
MORRILL, DR. A. D., Hamilton College, Clinton, New York.
NOYES, Miss EVA J.
PORTER, DR. H. C., University of Pennsylvania. Philadelphia, Pennsylvania.
SCOTT, DR. ERNEST L., Columbia University, New York City, New York.
SEARS, DR. HENRY F., 86 Beacon Street, Boston, Massachusetts.
SHEDD, MR. E. A.
THORNDIKE, DR. EDWARD L., Teachers College, Columbia University, New York
City, New York.
TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, New York.
TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, Illinois.
WAITE, PROF. F. C., 144 Locust Street, Dover, New Hampshire.
WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, California.
2. REGULAR MEMBERS
ABRAMOWITZ, DR. ALEXANDER A., Biological Laboratories, Harvard University,
Cambridge, Massachusetts.
ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts.
ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia.
Pennsylvania.
REPORT OF THE DIRECTOR
ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New
York.
ALBAUM, DR. HARRY G., 3115 Avenue I, Brooklyn, New York.
ALLEE, DR. W. C., The University of Chicago, Chicago, Illinois.
AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland,
School of Medicine, Lombard and Greene Streets, Baltimore, Maryland.
ANDERSON, DR. RUBERT S., Memorial Hospital, 444 East 68th Street, New York
City, New York.
ANGERER, DR. CLIFFORD A., Department of Physiology, Ohio State University, Co-
lumbus, Ohio.
ARMSTRONG, DR. PHILIP B., College of Medicine, Syracuse University, Syracuse,
New York.
AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts.
BAITSELL, DR. GEORGE A., Yale University, New Haven, Connecticut.
BAKER, DR. H. B., Zoological Laboratory, University of Pennsylvania, Philadelphia,
Pennsylvania.
BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire.
BALLENTINE, DR. ROBERT, Columbia University, Department of Zoology, New York
City, New York,
BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medi-
cal School, Boston, Massachusetts.
BARD, PROF. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland.
BARRON, DR. E. S. GUZMAN, Department of Medicine, The University of Chicago,
Chicago, Illinois.
BARTH, DR. L. G., Department of Zoology, Columbia University, New York City,
New York.
BEADLE, DR. G. \V., School of Biological Sciences, Stanford University, California.
BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, low^a
City, Iowa.
BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana.
BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge, Massachusetts.
BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cambridge, Massa-
chusetts.
BINFORD, PROF. RAYMOND, Buck Creek Camp, Marion, North Carolina.
BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut.
BLANCHARD, PROF. KENNETH C., Washington Square College, New York Univer-
sity, New York City, New York.
BODINE, DR. J. H., Department of Zoology, State University of Iowa, Iowa City,
Iowa.
BORING, DR. ALICE M., Yenching University, Peking, China.
BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wisconsin.
BRODIE, MR. DONALD M., 522 Fifth Avenue, New York City, New York.
BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, \Vashington Uni-
versity Medical School, St. Louis, Missouri.
BROOKS, DR. MATILDA M., University of California, Department of Zoology, Berke-
ley, California.
BROOKS. DR. S. C.. University of California, Berkeley, California.
26 MARINE BIOLOGICAL LABORATORY
BROWN, DR. DUGALD E. S., New York University, College of Dentistry, 209 East
23d Street, New York City, New York.
BROWN, DR. FRANK A., JR., Department of Zoology, Northwestern University,
Evanston, Illinois.
BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts.
BUCK, DR. JOHN B., Department of Zoology, University of Rochester, Rochester,
New York.
BUDINGTON, PROF. R. A., Winter Park, Florida.
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia.
BUMPUS, PROF. H. C., Duxbury, Massachusetts.
BYRNES, DR. ESTHER F., 1803 North Camac Street, Philadelphia, Pennsylvania.
CALKINS, PROF. GARY N., Columbia University, New York City, New York.
CANNAN, PROF. R. K., New York University College of Medicine, 477 First Ave-
nue, New York City, New York.
CARLSON, PROF. A. J., Department of Physiology, The University of Chicago, Chi-
cago, Illinois.
CAROTHERS, DR. E. ELEANOR, 134 Avenue C. East, Kingman, Kansas.
CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massachusetts.
CARROLL, PROF. MITCHELL, Franklin and Marshall College, Lancaster, Pennsyl-
vania.
CARVER, PROF. GAIL L., Mercer University, Macon, Georgia.
CATTELL, DR. McKEEN, Cornell University Medical College. 1300 York Avenue,
New York City, New York.
CATTELL, PROF. J. McKEEN, Garrison-on-Hudson, New York.
CATTELL, MR. WARE, Smithsonian Institution Building, Washington, D. C.
CHAMBERS, DR. ROBERT, Washington Square College, New York University, Wash-
ington Square, New York City, New York.
CHASE, DR. AURIN M., Princeton University, Princeton, New Jersey.
CHENEY, DR. RALPH H., Biology Department, Long Island University, Brooklyn,
New York.
CHIDESTER, PROF. F. E., Auburndale, Massachusetts.
CHILD, PROF. C. M., Jordan Hall, Stanford University, California.
CHURNEY, DR. LEON, 155 Powell Lane, Upper Darby, Pennsylvania.
CLAFF, MR. C. LLOYD, Department of Biology, Brown University, Providence,
Rhode Island.
CLARK, PROF. E. R., University of Pennsylvania Medical School, Philadelphia,
Pennsylvania.
CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, New
York.
CLELAND, PROF. RALPH E., Indiana University, Bloomington, Indiana.
CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana.
COE, PROF. W. R., Yale University, New Haven, Connecticut.
COHN, DR. EDWIN J., 183 Brattle Street, Cambridge, Massachusetts.
COLE, DR. ELBERT C., Department of Biology, Williams College, Williamstown,
Massachusetts.
COLE, DR. KENNETH S., College of Physicians and Surgeons, Columbia University,
630 West 168th Street, New York City, New York.
COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio.
REPORT OF THE DIRECTOR 27
COLTON, PROF. N. S., Box 601, Flagstaff, Arizona.
COOPER, DR. KENNETH W., Department of Biology, Princeton University, Prince-
ton, New Jersey.
COPELAND, PROF. MANTON, Bowdoin College, Brunswick, Maine.
COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina.
COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro-
lina, Chapel Hill, North Carolina.
CRAMPTON, PROF. H. E., Barnard College, Columbia University, New York City,
New York.
CROWELL, DR. P. S., JR., Department of Zoology, Miami University, Oxford, Ohio.
CURTIS, DR. MAYNIE R., 377 Dexter Trail, Mason, Michigan.
CURTIS, PROF. W. C., University of Missouri, Columbia, Missouri.
DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan.
DAVIS, DR. DONALD W., College of William and Mary, Williamsburg, Virginia.
DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts.
DAWSON, DR. J. A., The College of the City of New York, New York City, New
York.
DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut.
DEMEREC, DR. M., Carnegie Institution of Washington, Cold Spring Harbor, Long
Island, New York.
DILLER, DR. WILLIAM F., 1016 South 45th Street, Philadelphia, Pennsylvania.
DODDS, PROF. G. S., Medical School, University of West Virginia, Morgantown,
West Virginia.
DOLLEY, PROF. WILLIAM L., University of Buffalo, Buffalo, New York.
DONALDSON, DR. JOHN C., University of Pittsburgh, School of Medicine, Pitts-
burgh, Pennsylvania.
DuBois, DR. EUGENE F., Cornell University Medical College, 1300 York Avenue,
New York City, New York.
DUGGAR, DR. BENJAMIN M., University of Wisconsin, Madison, Wisconsin.
DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota.
DURYEE, DR. WILLIAM R., Department of Biology, Washington Square College,
New York University, New York City, New York.
EDWARDS, DR. D. J., Cornell University Medical College, 1300 York Avenue, New
York City, New York.
ELLIS, DR. F. W., Monson Massachusetts.
EVANS, DR. TITUS C., 723 Kirkwood, Iowa City, Iowa.
FAILLA, DR. G., College of Physicians and Surgeons, 630 West 168th Street, New
York City, New York.
FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France.
FERGUSON, DR. JAMES K. W., Department of Pharmacology, University of Toronto,
Ontario, Canada.
FIGGE, DR. F. H. J., 4636 Schenley Road, Baltimore, Maryland.
FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Rich-
mond, Virginia.
FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, To-
ronto, Canada.
MARINE BIOLOGICAL LABORATORY
FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto,
Canada.
FLEISHER, DR. MOVER S., 20 North Kingshighway, St. Louis, Missouri.
FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Massachu-
setts.
FRISCH, DR. JOHN A., Canisius College, Buffalo, New York.
FURTH, DR. JACOB, Cornell University Medical College, 1300 York Avenue, New
York City, New York.
GAGE, PROF. S. H., Cornell University, Ithaca, New York.
GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy Chase, Mary-
land.
GARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee.
GEISER, DR. S. W., Southern Methodist University, Dallas, Texas.
GERARD, PROF. R. W., The University of Chicago, Chicago, Illinois.
GLASER, PROF. O. C., Amherst College, Amherst, Massachusetts.
GOLDFORB, PROF. A. J., College of the City of New York, Convent Avenue and 139th
Street, New York City, New York.
GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut.
GOTTSCHALL, DR. GERTRUDE Y., 1630 Rhode Island Avenue, N.W., Washington,
D. C.
GRAHAM, DR. J. Y., University of Alabama, University, Alabama.
GRAND, CONSTANTINE G., Biology Department, Washington Square College, New
York University, Washington Square, New York City, New York.
GRAVE, PROF. B. H., DePauw University, Greencastle, Indiana.
GRAVE, PROF. CASWELL, Washington University, St. Louis, Missouri.
GRAY, PROF. IRVING E., Duke University, Durham, North Carolina.
GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City,
New York.
GUDERNATSCH, J. FREDRICK, New York University, 100 Washington Square, New
York City, New York.
GUTHRIE, DR. MARY J., University of Missouri, Columbia, Missouri.
GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin.
HAGUE, DR. FLORENCE, Sweet Briar College, Sweet Briar, Virginia.
HALL, PROF. FRANK G., Duke University, Durham, North Carolina.
HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St.
Louis, Missouri.
HANCE. DR. ROBERT T., Department of Biology, Duquesne University, Pittsburgh,
Pennsylvania.
HARGITT, PROF. GEORGE T., Department of Zoology, Duke University, Durham,
North Carolina.
HARMAN, DR. MARY T., Kansas State Agricultural College, Manhattan, Kansas.
HARNLY, DR. MORRIS H., Washington Square College, New York University, New
York City, New York.
HARPER, PROF. R. A., R. No. 5. Bedford, Virginia.
HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut.
HARTLINE, DR. H. KEFFER, University of Pennsylvania, Philadelphia, Pennsylvania.
HARTMAN, DR. FRANK A., Hamilton Hall. Ohio State University, Columbus, Ohio.
REPORT OF THE DIRECTOR
HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New Jer-
sey.
HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey.
HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachusetts.
HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie University, Halifax,
Nova Scotia.
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts.
HAZEN, DR. T. E., Barnard College, Columbia University, New York City, New
York.
HECHT, DR. SELIG, Columbia University, New York City, Ne\v York.
HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania.
HENDEE, DR. ESTHER CRISSEY, Russell Sage College, Troy, New York.
HENSHAW, DR. PAUL S., National Cancer Institute, Bethesda, Maryland.
HESS, PROF. WALTER N., Hamilton College, Clinton, New York.
HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio.
HILL, DR. SAMUEL E., Department of Biology. Russell Sage College, Troy, New
York.
HINRICHS, DR. MARIE, Department of Physiology and Health Education, South
Illinois Normal University, Carbondale, Illinois.
HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts.
HOADLEY, DR. LEIGH, Harvard University, Cambridge, Massachusetts.
HOBER, DR. RUDOLF, University of Pennsylvania, Philadelphia, Pennsylvania.
HODGE, DR. CHARLES, IV, Temple University, Department of Zoology, Philadelphia,
Pennsylvania.
HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia,
Pennsylvania.
HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Laboratory of In-
dustrial Hygiene, Bethesda, Maryland.
HOOKER, PROF. DAVENPORT, University of Pittsburgh, School of Medicine, Depart-
ment of Anatomy, Pittsburgh, Pennsylvania.
HOPKINS, DR. DWIGHT L., Mundelein College, 6363 Sheridan Road, Chicago, Illi-
nois.
HOPKINS, DR. HOYT S., New York University, College of Dentistry, New York-
City, New York.
HOWE,' DR. H. E., 1155 16th St., N.W., American Chemical Society Bldg., Wash-
ington, D. C.
HOWLAND, DR. RUTH B., Washington Square College, New York University.
Washington Square East, New York City, New York.
HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Virginia.
HYMAN, DR. LIBBIE H., American Museum of Natural History, New York City.
New York.
IRVING, PROF. LAURENCE, Swarthmore College, Swarthmore, Pennsylvania.
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts.
JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsylvania, Phila-
delphia, Pennsylvania.
JENKINS, DR. GEORGE B., 30 Gallatin Street, N.W., Washington, D. C.
30 MARINE BIOLOGICAL LABORATORY
JENNINGS, PROF. H. S., Department of Zoology, University of California, Los An-
geles, California.
JEWETT, PROF. J. R., 44 Francis Avenue, Cambridge, Massachusetts.
JOHLIN, DR. J. M., Vanderbilt University Medical School, Nashville, Tennessee.
JONES, DR. E. RUFFIN, JR., College of William and Mary, Williamsburg, Virginia.
KAUFMANN, PROF. B. P., Carnegie Institution, Cold Spring Harbor, Long Island,
New York.
KEMPTON, PROF. RUDOLF T., Vassar College, Poughkeepsie, New York.
KIDDER, DR. GEORGE W., Brown University, Providence, Rhode Island.
KILLE, DR. FRANK R., Swarthmore College. Swarthmore, Pennsylvania.
KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia.
KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, 36th Street and
Woodland Avenue, Philadelphia, Pennsylvania.
KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa.
KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York.
KOPAC, DR. M. J., Washington Square College, New York University, New York-
City, New York.
KORR, DR. I. M., Department of Physiology, New York University, College of Medi-
cine, 477 First Avenue, New York City, New York.
KRAHL, DR. M. E., Lilly Research Laboratories, Indianapolis, Indiana.
KRIEG, D*. WENDELL J. S., New York University, College of Medicine, 477 First
Avenue, New York City, New York.
LANCEFIELD, DR. D. E., Queens College, Flushing, New York.
LANCEFIELD, DR. REBECCA C, Rockefeller Institute, 66th Street and York Avenue,
Newr York City, New York.
LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts.
LEWIS, PROF. I. F., University of Virginia, Charlottesville, Virginia.
LILLIE, PROF. FRANK R., The University of Chicago, Chicago, Illinois.
LILLIE, PROF. RALPH S., The University of Chicago, Chicago, Illinois.
LOEB, PROF. LEO, 40 Crestwood Drive, St. Louis, Missouri.
LOEWI, PROF. OTTO, 155 East 93d Street, New York City, New York.
LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York
City, New York.
LUCAS, DR. ALFRED M., Zoological Laboratory, Iowa State College, Ames, Iowa.
LUCAS, DR. MIRIAM SCOTT, Department of Zoology, Iowa State College, Ames,
Iowa.
LUCRE, PROF. BALDUIN, University of Pennsylvania, Philadelphia, Pennsylvania.
LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Avenue, New
York City, New York.
LYNCH, DR. RUTH STOCKING, Maryland State Teachers College, Towson, Mary-
land.
LYNN, DR. WILLIAM G., Department of Biology, The Catholic University of Amer-
ica, Washington, D. C.
MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia.
MACLENNAN, DR. RONALD F., 174 Forest Street, Oberlin, Ohio.
MACNAUGHT, MR. FRANK M., Marine Biological Laboratory, Woods Hole, Massa-
chusetts.
REPORT OF THE DIRECTOR 31
McCLUNG, PROF. C. E., 417 Harvard Avenue, Swarthmore, Pennsylvania.
McCoucH, DR. MARGARET SUM WALT, University of Pennsylvania Medical School,
Philadelphia, Pa.
MCGREGOR, DR. J. H., Columbia University, New York City, New York.
MACKLIN, DR. CHARLES C., School of Medicine, University of Western Ontario,
London, Canada.
MAGRUDER. DR. SAMUEL R., Department of Anatomy, Tufts Medical School, Bos-
ton, Massachusetts.
MALONE, PROF. E. F., College of Medicine, University of Cincinnati, Department
of Anatomy, Cincinnati, Ohio.
MAN WELL, DR. REGINALD D., Syracuse University, Syracuse, New York.
MARSLAND, DR. DOUGLAS A., Washington Square College, New York University,
Xew York City, New York.
MARTIN, PROF. E. A., Department of Biology, Brooklyn College, Bedford Avenue
and Avenue H, Brooklyn, New York.
MAST, PROF. S. O., Johns Hopkins University, Baltimore, Maryland.
MATHEWS, PROF. A. P., University of Cincinnati, Cincinnati, Ohio.
MATTHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College,
Williamstown, Massachusetts.
MAYOR, PROF. JAMES W., Union College, Schenectady, New York.
MAZIA, DR. DANIEL, Department of Zoology, University of Missouri, Columbia,
Missouri.
MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania.
MEIGS, MRS. E. B., 1736 M Street, N.W., Washington. D. C.
MENKIN. DR. VALY, Harvard Medical School, Boston, Massachusetts.
METZ, PROF. CHARLES W., University of Pennsylvania, Philadelphia, Pennsylvania.
MICHAELIS, DR. LEONOR, Rockefeller Institute, 66th Street and York Avenue, New
York City, New York.
MILLER, DR. J. A., Division of Anatomy, College of Medicine, University of Ten-
nessee, Memphis, Tennessee.
MINNICH, PROF. D. F., Department of Zoology, University of Minnesota, Minne-
apolis, Minnesota.
MITCHELL, DR.' PHILIP H., Brown University, Providence, Rhode Island.
MOORE, DR. CARL R., The University of Chicago, Chicago, Illinois.
MORGAN, DR. ISABEL M., Rockefeller Institute, York Avenue at 66th Street, New
York City, New York.
MORGULIS, DR. SERGIUS, University of Nebraska, Omaha, Nebraska.
MORRILL, PROF. C. V., Cornell University Medical College, 1300 York Avenue,
New York City, New York.
MOSER, DR. FLOYD, Department of Biology, University of Alabama, University,
Alabama.
MULLER, PROF. H. J., Amherst College, Amherst, Massachusetts.
NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Milton, Massa-
chusetts.
NEWMAN, PROF. H. H., 173 Devon Drive, Clearwater, Florida.
NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania.
NONIDEZ. DR. JOSE F., Cornell University Medical College, 1300 York Avenue,
New York City, New York.
32 MARINE BIOLOGICAL LABORATORY
NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New Jersey.
OKKELBERG, DR. PETER, Department of Zoology, University of Michigan, Ann
Arbor, Michigan.
OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn
Mawr, Pennsylvania.
OSBURN, PROF. R. C, Ohio State University, Columbus, Ohio.
OSTERHOUT, PROF. W. J. V., Rockefeller Institute, 66th Street and York Avenue,
New York City, New York.
OSTERHOUT, MRS. MARIAN IRWIN, Rockefeller Institute, 66th Street and York
Avenue, New York City, New York.
PACKARD, DR. CHARLES, Marine Biological Laboratory, Woods Hole, Massachu-
setts.
PAGE, DR. IRVINE H., Lilly Laboratory Clinical Research, Indianapolis City Hos-
pital, Indianapolis, Indiana.
PAPPENHEIMER, DR. A. M., Columbia University, New York City, New York.
PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts.
PARMENTER, DR. C. L., Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania.
PARPART, DR. ARTHUR K., Princeton University, Princeton, New Jersey.
PATTEN, DR. BRADLEY M., University of Michigan Medical School. Ann Arbor.
Michigan.
PAYNE, PROF. F., University of Indiana, Bloomington, Indiana.
PEEBLES, PROF. FLORENCE, Lewis and Clark College, Portland, Oregon.
PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wisconsin.
PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts.
POLLISTER, DR. A. W., Columbia University, New York City, New York.
POND, DR. SAMUEL E., 1203 Enfield Street, Thompsonville, Connecticut.
PRATT, DR. FREDERICK H., Boston University, School of Medicine, Boston, Massa-
chusetts.
PROSSER, DR. C. LADD, University of Illinois, Urbana, Illinois.
RAND, DR. HERBERT W., Harvard University, Cambridge, Massachusetts.
RANKIN, DR. JOHN S., Zoology Department, University of Washington, Seattle,
Washington.
REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massachusetts.
RENSHAW, PROF. BIRDSEY, 4600 Harling Lane, Bethesda, Maryland.
DERENYI, DR. GEORGE S., Department of Anatomy, University of Pennsylvania,
Philadelphia, Pennsylvania.
REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue,
New York City, New York.
RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio.
RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma.
RICHARDS, PROF. A. G., Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania.
RICHARDS, DR. O. W., Research Department, Spencer Lens Company, 19 Doat
Street, Buffalo, New York.
RIGGS, LAWRASON, JR., 120 Broadway, New York City, New York.
ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio.
ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts.
REPORT OF THE DIRECTOR
ROOT, DR. R. W., Department of Biology, College of the City of New York, Con-
vent Avenue and 139th Street, New York City, New York.
ROOT, DR. W. S., College of Physicians and Surgeons, Department of Physiology,
630 West 168th Street, New York City, New York.
RUEBUSH, DR. T. K., Naval Medical School, National Naval Medical Center, Beth-
esda, Maryland.
RUGH, DR. ROBERTS, Department of Biology, Washington Square College, New
York University, New York City, New York.
SASLOW, DR. GEORGE, 72 Grozier Road, Cambridge, Massachusetts.
SAYLES, DR. LEONARD P., Department of Biology, College of the City of New York,
139th Street and Convent Avenue, New York City, New York.
SCHAEFFER, DR. ASA A., Biology Department, Temple University, Philadelphia,
Pennsylvania.
SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street and Con-
vent Avenue, New York City, New York.
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio.
SCHMITT, PROF. F. O., Department of Biology and Public Health, Massachusetts
Institute of Technology, Cambridge, Massachusetts.
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massa-
chusetts.
SCHRADER, DR. FRANZ, Department of Zoology, Columbia University, New York
City, New York.
SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia University, New
York City, New York.
SCHRAMM, PROF. J. R., University of Pennsylvania, Philadelphia, Pennsylvania.
SCOTT, DR. ALLAN C.. Union College, Schenectady, New York.
SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, New Jersey.
SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College, Greens-
burg, Pennsylvania.
SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New York.
SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and
Surgeons, 630 West 168th Street, New York City, New York.
SHAPIRO, DR. HERBERT, Radiation Laboratory, Massachusetts Institute of Technol-
ogy, Cambridge, Massachusetts.
SHELFORD, PROF. V. E., Vivarium, Wright and Healey Streets, Champaign, Illinois.
SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Michigan.
SHUMWAY, DR. WALDO, University of Illinois, Urbana, Illinois.
SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont.
SICHEL, MRS. F. J. M., 35 Henderson Terrace, Burlington, Vermont.
SINNOTT, DR. E. W., Osborn Botanical Laboratory, Yale University, New Haven,
Connecticut.
SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa
City, Iowa.
SMITH, DR. DIETRICH CONRAD, Department of Physiology, University of Mary-
land School of Medicine, Lombard and Greene Streets, Baltimore, Maryland.
SNYDER, PROF. L. H., Ohio State University, Department of Zoology, Columbus.
Ohio.
SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio.
34 MARINE BIOLOGICAL LABORATORY
SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington,
Indiana.
SPEIDEL, DR. CARL C., University of Virginia. University, Virginia.
STABLER, DR. ROBERT M., Department of Zoology, University of Pennsylvania,
Philadelphia, Pennsylvania.
STARK, DR. MARY B., 1 East 105th Street, New York City, New York.
STEINBACH, DR. H. BURR, Department of Zoology, Washington University, St.
Louis, Missouri.
STERN, DR. CURT, Department of Zoology, University of Rochester, Rochester,
New York.
STERN, DR. KURT G., Overly Biochemical Research Foundation, 254 W. 31st
Street, New York City, New York.
STEWART, DR. DOROTHY R., Skidmore College, Saratoga Springs, New York.
STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College, South
Hadley, Massachusetts.
STRONG, PROF. O. S., College of Physicians and Surgeons, Columbia University,
New York City, New York.
STUNKARD, DR. HORACE W., New York University, University Heights, New
York.
STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena,
California.
SUMMERS, DR. FRANCIS MARION. Department of Biology, College of the City of
New York. New York City, New York.
SWETT, DR. FRANCIS H., Duke University Medical School, Durham, North
Carolina.
TAFT, DR. CHARLES H., JR., University of Texas Medical School, Galveston, Texas.
TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati, Ohio.
TAYLOR, DR. C. V., Leland Stanford University, Leland Stanford, California.
TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michigan.
TEWINKEL, DR. L. E.. Department of Zoology, Smith College, Northampton,
Massachusetts.
TURNER, DR. ABBY H., Department of Physiology, Mount Holyoke College, South
Hadley, Massachusetts.
TURNER. PROF. C. L., Northwestern University, Evanston, Illinois.
TYLER, DR. ALBERT, California Institute of Technology, Pasadena, California.
UHLENHUTH, DR. EDUARD, University of Maryland, School of Medicine, Balti-
more, Maryland.
UNGER, DR. W. BYERS, Dartmouth College, Hanover, New Hampshire.
VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland, Ohio.
WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge,
Massachusetts.
WARD, PROF. HENRY B., 1201 W. Nevada, Urbana, Illinois..
WARREN, DR. HERBERT S., 1405 Greywall Lane, Overbrook Hills, Pennsylvania.
WATERMAN, DR. ALLYN J., Department of Biology, Williams College, Williams-
town, Massachusetts.
WEISS, DR. PAUL A., Department of Zoology, The University of Chicago, Chicago,
Illinois.
WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pennsylvania.
REPORT OF THE DIRECTOR
WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, North Dakota.
WHITAKER, DR. DOUGLAS M., P. O. Box 2514, Stanford University, California.
WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania.
WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania,
Philadelphia, Pennsylvania.
WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Nebraska.
WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia,
Pennsylvania.
WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, Ohio.
WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore,
Maryland.
WILSON, DR. J. W., Brown University, Providence, Rhode Island.
WITSCHI, PROF. EMIL, Department of Zoology, State University of Iowa, Iowa
City, Iowa.
WOLF, DR. ERNST, Biological Laboratories, Harvard University, Cambridge,
Massachusetts.
WOODRUFF, PROF. L. L., Yale University, New Haven, Connecticut.
WOODWARD, DR. ALVALYN E., Zoology Department, University of Michigan, Ann
Arbor, Michigan.
YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medical College,
1300 York Avenue, New York City, New York.
YOUNG, DR. B. P., Cornell University, Ithaca, New York.
YOUNG, DR. D. B.. 7128 Hampden Lane, Bethesda, Maryland.
SEXUAL ISOLATION, MATING TYPES, AND SEXUAL RESPONSES TO
DIVERSE CONDITIONS IN VARIETY 4, PARAMECIUM AURELIA l
T. M. SONNEBORN 2 AND RUTH V. DIPPELL
(Department of Zoology, Indiana University, Bloomingtori)
In previous publications (Sonneborn, 1938; 1939; 1943) the species Para-
rnecium aurelia has been shown to consist of a number of sexually isolated
and physiologically distinct groups of races. Their sexual isolation is perhaps
sufficient ground for assigning these groups to different species; but as all are
morphologically similar and conform to the description of the species Paramecium
aurelia, it seems more practical for the present at least to designate them as
varieties of this species. Each of these varieties consists of two classes of in-
dividuals that are morphologically identical but physiologically different. These
two classes of individuals mate with each other, but neither class mates with other
individuals of the same class or with either of the two classes that occur in any
other variety of the species. The two classes of individuals within each variety
are known as mating types and, in P. aurelia, they are designated by Roman
numerals. The diverse varieties are designated by Arabic numerals.
The present paper is the first of a series dealing with the general biology and
genetics of variety 4, containing the mating types VII and VIII. Each variety
thus far studied has proven to be specially favorable for the study of certain
problems of protozoan biology and genetics not so readily investigated in other
varieties. As will appear in the course of this series of papers, investigations on
variety 4 have yielded information on a number of important problems. In this
first paper of the series we set forth the foundation on which the work of the later
papers is based : demonstration of the existence of variety 4, and an account of its
mating types and the conditions under which they mate.
MATERIAL
Among the 53 races of P. aurelia collected from different sources in nature and
studied in this laboratory, only the following four belong to variety 4:
Race 29 collected by Dr. R. F. Kimball from Ben's Run, Hebbville, Maryland, in
1938.
Race 32 collected by Dr. Kimball from a pond in Towson, Maryland, in 1938.
Race 47 collected by Dr. A. C. Giese from a pool across the Bay from Berkeley,
California, and sent to me in February 1939.
Race 51 collected by Mrs. Aner Laubscher at Spencer, Indiana, in August 1939.
Before intensive study of these races began in the spring of 1942, they were
maintained in quart jars of hay infusion to which boiled hay strips were added
every month or two. In the course of this period, race 47 either changed one
1 Contribution No. 318 from the Department of Zoology, Indiana University.
2 Aided by a grant from the Rockefeller Foundation.
36
MATING TYPES IN PARAMECIUM AURELIA 37
of its characters or was mislabelled, for in 1939 it produced a unique type of
lethal action on other races and no trace of this action has appeared in our recent
work. In the following studies, these four races were cultivated in desiccated
lettuce infusion to which a pure culture of the bacterium Aerobacter aerogenes
was added.
OCCURRENCE, SEXUAL ISOLATION AND MATING TYPES OF VARIETY 4
In order to discover whether a race or group of races constitutes a new variety
(in the sense in which this term is employed here, i.e., a sexually isolated group
of races), it is required to demonstrate that it contains mating types which inter-
breed with each other but not with those in any other known variety. This is
made possible by the fact that all the mating types so far found in P. aurelia
ordinarily reproduce true to type during vegetative reproduction and so yield
from a single individual a clone containing one mating type only. Samples of
clones of unidentified races may then be mixed with samples of sexually reactive
clones of each of the known mating types. If no mating occurs in any of these
mixtures, this is evidence that the new races do not contain any of the known
mating types; but the evidence is not convincing unless it is certain that the clones
of the new races, as well as those of the known mating types, are in sexually
reactive condition at the time the tests are carried out. This can be achieved
only when the clones of the new races mate with each other in appropriate
combinations.
Such an analysis was carried out on the four races discusssed in this paper.
Clones of each of these races were mixed with sexually reactive cultures of each
of the six known mating types (I, II, III, IV, V, and VI) and no mating resulted.
As repeated trials gave the same result, the six known mating types seemed not
to occur among the four new races. However, at that time mating also failed
to occur in mixtures of different clones and different races of the four new races.
Under such conditions, conclusive proof that they constituted a new variety could
not be given; they might simply have been immature. In April 1942 this diffi-
culty disappeared when mating was observed for the first time in race 32. As
some of the individuals were coming together in preparation for conjugation,
they were separated before they had time to unite firmly and cultures were grown
from the isolated members of the split pairs. The resulting clones proved to
be of unlike mating types for no mating occurred within either clone alone, but
the characteristic clumping reaction and conjugation took place when samples of
the two clones from a split pair were mixed together. The same clones, while in
this reactive condition, failed to clump or conjugate with any of the six pre-
viously known mating types, although all of these were at the time in highly
reactive sexual condition. Hence, there occur in race 32 two mating types unlike
any of those previously known. They were therefore called mating types VII
and VIII. All clones of race 32 available at that time, and subsequently, have
been found to belong to either one or the other of these two mating types. When
these two types were mixed with samples of clones of the remaining three races
(29, 47 and 51), clumping and conjugation occurred in the mixtures with type:
VIII, but not in the mixtures with type VII. These three races therefore con-
tained type VII only and all clones examined at that time in these three races
38 SONNEBORN AND DIPPELL
were found to be of type VII. The four races 29, 32, 47 and 51 thus constitute
a fourth variety with two new mating types VII and VIII.
Subsequently, and at a definitely known time, type VIII arose independently
in race 51, but it has still not been found in races 29 or 47 in spite of a prolonged
and intensive search for it. However, type VIII might well arise eventually in
these races also as it has already done in the other two races.
SEXUAL RESPONSES TO DIVERSE CONDITIONS IN VARIETY 4
The nutritive conditions for conjugation appear to be the same in variety 4
as in the three previously described varieties: the animals must be neither very
well fed nor completely starved, but in a declining nutritive condition. The
strongest mating reactions take place when there are in progress, in the cultures
to be mixed, the last fissions before the food supply is exhausted.
As diurnal periodicities in the occurrence of the mating reaction exist in two
of the three previously described varieties of P. aurelia (Sonneborn, 1938; 1939),
the possibility of its occurrence was examined in variety 4. For this purpose,
cultures of the races 29 and 47 and cultures of each mating type in the races 32
and 51 were prepared by growing them for 6 days exposed to the light of a north
window during the daylight hours. The plan was to mix samples of each of the
type VIII cultures (from races 32 and 51) with each of the type VII cultures (from
all four of the races) at four-hour intervals through at least one complete cycle
of 24 hours. In order to be sure to have cultures in the proper nutritive condi-
tion at all times, the six original cultures were subcultured in triplicate the evening
before the tests were to be made and the three subcultures of each original were
fed in the ratio of 1 : 2 : 4 volumes of culture fluid. During the daylight hours
there was no difficulty in making the required mixtures, but at night precautions
had to be taken to avoid exposing the cultures to light in so far as possible. This
was accomplished as follows. Samples of all the cultures to be mixed at night
were put into depression slides before dark. The two depressions of each slide
contained two cultures that were later to be mixed. There was a separate slide
for each combination and each time of mixture, with ample duplicates for emer-
gencies. All of these slides were placed in moist chambers and were covered at
night with black cloth. At the time for mixture, a very dim flashlight was di-
rected away from the culture dishes, the appropriate slides were removed from
the moist chambers, and the fluid from one depression on each slide was pipetted
into the other depression of the same slide. Two or three minutes later the mix-
ture was examined under the microscope with the faint light from the flashlight.
The mixtures were then returned to the cloth-covered moist chambers.
A complete set of eight mixtures was made every four hours beginning at
5:15 P.M. on February 13 and continuing until 9:15 P.M. on February 14
Additional sets were made on other days at various times from 8 A.M. to 10 P.M.
The agglutinative mating reaction occurred at once in mixtures made at every
one of the different hours tested. There was thus no indication of any diurnal
periodicity in the mating reaction. In this respect variety 4 is like variety 1
and unlike varieties 2 and 3 (Sonneborn, 1938; 1939).
The relation of temperature to the occurrence of conjugation was studied in
five series of experiments. In each series, the same eight combinations of cul-
MATING TYPES IN PARAMECIUM AURELIA 39
tures were brought together as in the preceding experiments on diurnal periodicity.
In series 1, each of the six cultures was grown for 6 days at 9°, 16.5°, 20° and 25° C. ;
then a set of eight mixtures wras made and retained at each temperature and
duplicate sets from 9° and 16.5° were immediately placed at 25°. In series 2,
the same cultures were grown for one day at 9°, 15.5°, 21° and 25.5°; mixtures
were made as in series 1, duplicate sets of mixtures from the two lower tempera-
tures again being placed at once at the highest temperature. In series 3, the same
six cultures were grown for 13 days at 9°, 15.5° and 26°; then mixtures were made
and retained at the same temperatures and duplicate sets of mixtures from the
two lower temperatures were again placed at the highest temperature; in addi-
tion two extra sets of mixtures were made from the 26° cultures: one was im-
mediately placed at 9° and the other at 15.5°. In series 4, cultures were grown
for one day at 22°, 30° and 36°; one set of mixtures was made and retained at each
temperature, one set from 30° and one from 36° was placed at 22° and two sets
from 22° were placed at 30° and 36° respectively. In series 5 the six cultures
were grown for several days at 21°, then five sets of mixtures were placed at
10°, 19°, 24.5°, 29° and 39°, respectively. We report first the results on mixtures
retained at the temperatures at which the cultures were grown, then the results of
changing the temperature at the time the mixtures were made.
Cultures Grown and Tested at 9° C. Three sets of eight mixtures between
types VII and VIII (series 1, 2, and 3) were grown and tested at 9°. In 20 of
these mixtures no conjugation occurred at all; in the other four mixtures (all from
series 1) less than 3 per cent of the animals conjugated. The mixtures of series
1 were observed 8^ hours; series 2, 31 hours; and series 3, 23 days. Thus at 9°
conjugation occurs in but a small proportion of mixtures and among only small
proportions of the animals in these.
Cultures Grown and Tested at 15.5° to 16.5° C. Three sets (series 1,2, and 3)
of eight mixtures each were grown and tested at this temperature. The first
two sets reacted poorly: half of the 16 mixtures gave no conjugation at all and
the other half gave only 1 to 3 per cent conjugation. In the third set, one mixture
gave 50 per cent conjugation and the other seven gave 15 to 25 per cent. Thus
conjugation occurs in more of the cultures and may occur in a much higher pro-
portion of the animals of a culture at this temperature than at 9°.
Cultures Grown and Tested at 20° to 22° The 24 mixtures (series 1, 2 and 4)
grown and tested at this temperature all gave large proportions of conjugants —
30 per cent to 90 per cent — and most of them gave immediate strong agglutinative
reactions at the time of mixture. The latter did not occur at all at the lower
temperatures.
Cultures Grown and Tested at 25° to 26°. Of the 24 mixtures made at this
temperature, four proved unsuitable for study. The remaining 20 gave 40 per
cent to 90 per cent conjugation and most gave strong immediate agglutinative
mating reactions at the time of mixture.
Cultures Grown and Tested at 30°. The eight mixtures (series 4) at this
temperature all gave immediate strong mating reactions and high percentages
of conjugants.
Cultures Grown and Tested at 36°. The eight mixtures at this temperature
(series 4) gave from 2 to 20 per cent conjugation.
At 39° cultures could not be grown, but the effects of this temperature, as set
40 SONNEBORN AND DIPPELL
forth below, were studied in cultures grown at lower temperatures and placed
at 39° immediately after mixture.
From the preceding, it appears that the optimal temperatures for conjuga-
tion in variety 4 extend from 20° to 30°; that the amount of conjugation obtained
is approximately the same throughout this range of temperature; that the amount
decreases both as temperature rises and falls away from this range; and that it
occurs but rarely at 9°.
In the following paragraphs are presented the results of changing temperature
at the time cultures of types VII and VIII are mixed together. The changes of
temperature investigated were: (a) changes within the optimal range (20° to 30°);
(6) changes from optimal to non-optimal temperatures; and (c) changes from
non-optimal to optimal temperatures. The results, which are presented in this
order, confirm and extend the conclusions in the preceding paragraph concerning
the relation of temperature to the occurrence of conjugation in variety 4.
Changes of Temperature within the Optimal Range (20° to 30°). The following
changes of temperature within the optimal range were investigated: cultures
grown at 21°-22° were placed at the time of mixture at 24.5° (series 5), at 29°
(series 5), and at 30° (series 4) ; and cultures grown at 30° were placed at the time
of mixture at 22° (series 4). In each experiment, as in all of those that follow,
a complete set of eight mixtures was again made in the way set forth in the pre-
ceding section. After all of these changes of temperature, the proportions of
conjugants obtained in the mixtures were not significantly different from those
obtained in other mixtures of the same cultures kept at the original temperatures.
Hence, change of temperature within the optimal range has no effect on the pro-
portion of conjugants obtained.
Changes from Optimal to Non-optimal Temperatures. When cultures of the
two mating types were grown at a temperature within the range 20° to 30°, were
mixed together and placed immediately at a temperature well outside this range,
the proportions of animals that conjugated were always less than in corresponding
controls retained after mixture at the original temperature.
In two experiments the temperature was raised from 21° or 22° to well over
30°. In one experiment, increase of temperature from 22° to 36° (series 4) re-
sulted in no conjugation at all in two of the mixtures and in less than 12 per cent
conjugation in the other six mixtures. The corresponding control mixtures
retained at 22° gave in each of the eight mixtures from 30 to 90 per cent conjuga-
tion, or seven to eight times as much as in those placed at 36°. In the other
experiment, increase of temperature from 21° to 39° resulted in no conjugation at
all in any of the eight mixtures; but the corresponding eight control mixtures re-
tained at 21° all conjugated in high proportions. Hence the upper limit of
temperature for the occurrence of conjugation in variety 4 lies between 36°
and 39°.
The temperature was lowered from 21° or 26° to well below 20° in three experi-
ments. In one (series 5) the temperature was reduced from 21° to 10°. After
2 hours, the eight mixtures at 10° had less than half as many pairs of "conjugants"
as the eight control mixtures retained at 21°. Moreover, while the pairs in the
21° mixtures were tightly united, those in the 10° mixtures were not. As will
appear immediately, there is reason to believe that all of the latter pairs would
have separated without having conjugated. Evidence for this was obtained in
MATING TYPES IN PARAMECIUM AURELIA 41
the second experiment (series 3) in which the temperature was reduced from 26°
to 9°. Each of the eight control mixtures retained at 26° yielded more than 50
per cent of the animals tightly united in conjugation within 4 hours; but the eight
mixtures at 9° contained at this time less than 10 per cent of the animals in pairs
and these pairs were still loosely united. Soon thereafter all these pairs broke
apart without having united in true conjugation and no other pairs formed, even
loosely, within the next four days (compare with variety 1, Sonneborn, 1941).
Reduction of temperature from over 20° to 10° or less thus suppresses conjuga-
tion just as does an increase of temperature to 39°. The third 'experiment (series
3) involved reduction of temperature from 26° to 15.5°. These eight mixtures
each gave from 15 to 20 per cent conjugation, while each of the corresponding
control mixtures at 26° gave more than 50 per cent conjugation within four hours.
All five of these experiments agree in showing that change from a temperature
of 21° to 26° to one well below 20° or well above 30° results invariably in consider-
able reduction in the proportion of animals that conjugate. When the new tem-
perature is as low as 10° or as high as 39°, conjugation is completely suppressed.
Changes from Non-optimal to Optimal Temperatures. Such changes include
both reductions from very high to moderate temperatures and increases from
very low to moderate temperatures. Both types of changes resulted in increases
in the amount of conjugation. Thus, eight mixtures of cultures grown at 36° and
placed immediately at 22° gave 10 to 70 per cent conjugation in 6£ hours, while
corresponding control mixtures retained at 36° gave only 2 to 20 per cent con-
jugation in the same time. Further, three sets of cultures grown at 9° were mixed
and placed at 25°-26°. All 24 of these mixtures yielded conjugants in proportions
varying from 10 to 90 per cent; but 20 of the 24 control mixtures retained at 9°
yielded no conjugants at all and the other four gave less than 3 per cent conjuga-
tion. Finally, three sets of cultures grown at 15.5°-16.5° were mixed and placed
at 25°-26°. All 24 of these mixtures conjugated and gave higher proportions of
conjugants than the corresponding controls kept at 15.5°-16.5°. For example,
in one set, seven of the mixtures yielded 40 to 65 per cent conjugants while the
corresponding controls yielded only 15 to 25 per cent; and the eighth mixture
gave 75 per cent conjugation, its control only 50 per cent.
In general, when the temperature is changed at the time cultures of diverse
mating type are mixed, the percentage of conjugation that results is unaffected
if both the original and final temperatures are moderate (20° to 30°) ; it is greatly
increased if the original temperature is extreme (36° and above, or 16° and below)
and the final temperature moderate; and it is greatly decreased if the original
temperature is moderate and the final temperature extreme. The optimal tem-
peratures for the occurrence of conjugation in variety 4 are thus moderate (be-
tween 20° and 30°), regardless of whether mixtures are made from cultures grown
at these or other temperatures. Conversely, as the temperature at which the
mixtures are placed diverges from this optimum range (either above it or below) ,
the percentage of conjugation decreases.
DISCUSSION
The conditions for conjugation in variety 4 differ markedly from those for
varieties 2 and 3 in the same ways that the conditions for conjugation in variety 1
42 SONNEBORN AND DIPPELL
do (Sonneborn, 1938; 1939). Both varieties 1 and 4 lack a diurnal periodicity in
sexual reactivity. Both are able to conjugate over a wide range of tempera-
tures. Both give smaller proportions of conjugants as temperature decreases
below 20°. Both react to a sudden reduction of the temperature to 10° by dis-
continuing a mating reaction previously begun. Both are occasionally able to
conjugate at this low temperature, if cultures of opposite types are grown at the
same temperature some time before mixture. Nevertheless, varieties 1 and 4 do
differ slightly in the conditions for conjugation; but the differences appear only
at higher temperatures. Variety 4 gives maximum mating reactions between
20° and 30°, weak ones at 36° and fails to conjugate at 39°. Variety 1 gives maxi-
mum reactions between 20° and 38° and then suddenly fails to conjugate as the
temperature rises to 40°. Thus, although conjugation occurs over practically
the same range of temperature in the two varieties, the range of temperature for
maximum sexual reactivity and the rate at which sexual reactivity decreases as
the temperature rises above the optimum differ in the two varieties. At 36°
the difference appears clearly: variety 1 gives a maximum reaction, while variety
4 conjugates but poorly. Thus it is possible to distinguish these four varieties
of P. aurelia not only by their mating types, but also by the sexual responses to
diverse conditions. Whether the latter will hold for all varieties of P. aurelia
remains to be discovered. Four more varieties are under cultivation (reported
in part in Sonneborn, 1943) in our laboratory and many more must exist in
nature; but the sexual responses of these to diverse conditions have not yet been
investigated.
SUMMARY
Among the 53 races of P. aurelia that have been investigated, four races
(29, 32, 47 and 51) do not conjugate with any of the three previously described
varieties. They constitute a fourth variety with two new interbreeding mating
types, VII and VIII. Mating type VII occurs in all four of these races, but mat-
ing type VIII has appeared only in the two races 32 and 51.
The mating types VII and VIII give with each other the agglutinative mating
reaction characteristic of Paramecium and proceed to conjugate. As in the other
three varieties, agglutination and conjugation occur only when mixture is made
between cultures of the two types that are neither well-fed nor starved, but are
nearing the stage of nutritive exhaustion. Like variety 1, but unlike varieties
2 and 3, variety 4 shows no diurnal periodicity in sexual reactivity: cultures ex-
posed to the natural alternation of daylight and night are capable of reacting
sexually at any hour. Further, again like variety 1 and unlike varieties 2 and 3,
variety 4 can react sexually throughout the range of temperatures from 9° to
36°, but not at 39°. At 16°, the sexual reactions are weak, leading to but a small
proportion of conjugants. In mixtures made at higher temperatures and trans-
ferred at once to 9°, pairs begin to form but break apart without conjugating;
however, if cultures are first adapted to 9° before they are mixed, a small propor-
tion of true conjugation may occur at this temperature. In all these details,
varieties 1 and 4 are alike; but they differ in behavior at the higher temperatures.
The maximum optimum temperature for conjugation lies between 30° and 36° in
variety 4, between 38° and 40° in variety 1. Thus at 36°, variety 1 gives a
maximum sexual reaction, while variety 4 gives only 12 to 25 per cent of the
MATING TYPES IN PARAMECIUM AURELIA 43
optimum. Variety 4 shows a gradual falling off in sexual reactivity as tempera-
ture increases above the optimum, while variety 1 shows a sudden cessation of
sexual reactivity at a temperature only 2° above the optimum.
It is thus possible to distinguish these four varieties of P. aurelia not only by
their mating types, but also by the sexual responses to diverse conditions.
LITERATURE CITED
SONNEBORN, T. M., 1938. Mating types in Paramecium aurelia: diverse conditions for mating in
different stocks; occurrence, number and interrelations of the types. Proc. Amer. Phil.
Soc., 79: 411-434.
SONNEBORN, T. M., 1939. Paramecium aurelia: mating types and groups; lethal interactions;
determination and inheritance. Amer. Nat., 73: 390-413.
SONNEBORN, T. M., 1941. The effect of temperature on mating reactivity in Paramecium aurelia,
variety 1. Anat. Rec. 81 (suppl): 131.
SONNEBORN, T. M., 1943. More mating types and varieties in Paramecium aurelia. Anat. Rec.,
84(4): 92.
HYBRIDIZATION AND SEASONAL SEGREGATION IN
TWO RACES OF A BUTTERFLY OCCURRING
TOGETHER IN TWO LOCALITIES
WILLIAM HOVANITZ
(California Institute of Technology, Pasadena) %
The yellow and orange butterfly, Colias chrysotheme, exists in the form of two
complexes known as the orange-race and the yellow-race (Hovanitz, 1943a;
1943b). These races have different geographical distributions but overlap over
a tremendous territory from the Sierra-Cascade divide in western North America
to the Atlantic ocean in the east and from southern Canada in the north through
Mexico in the south (Hovanitz, 1943c). Each race usually occupies a different
ecologic niche so that nearly pure populations of each may be found in this area
as well as outside the zone of overlap. In certain localities, however, the same
ecologic niche is partly occupied by both races, resulting in considerable hy-
bridization between them.
Two localities where the races occupy the same niche for the most part were
analyzed from 1941 to 1943 in order to study the behavior of each in relation to
its environment, and to get an indication of the extent of hybridization between
them. These places were at Mono Lake Valley, Mono County, California, and
at Round Valley (near Bishop), Inyo County, California. Their positions are
indicated on a map (Hovanitz, 1943d) ; they are just east of the Sierra Nevada in
the western Great Basin.
The Seasonal Distribution of Adults
Orange butterflies are present throughout the entire warm season of the year
at both Round Valley and Mono Lake. It is easier, however, to get a good sample
in midsummer as compared with early spring or autumn. The abundance of
orange adults apparently is at a minimum at each end of the growing season and
at a maximum in midsummer.
The yellow butterflies at Mono Lake are more irregular in seasonal distribu-
tion than the orange (Fig. 1). The 1941 samples (Table I) show a high relative
frequency of yellow to orange in May, and then a complete drop to none present
at all in June. A rise to a second maximum in late July is apparent with a
gradual drop again to none at all in September. Early in October there is a
third maximum. This suggests three distinct broods per year at Mono Lake with
an elapsed egg, larval and pupal development time of two months between each.
This time compares with a development rate of three to four weeks at a constant
laboratory temperature of 25° C. Mono Lake has a rather low air temperature,
especially at night; in the day time, the direct radiation from the sun is the
primary source of heat.
The 1942 samples at Mono Lake show much the same seasonal distribution.
44
HYBRIDIZATION IN BUTTERFLIES 45
The first adult flight was apparently not observed; it is probably very short in
duration. The 1942 samples were obtained at monthly intervals rather than semi-
monthly as in 1941; therefore, the chance of missing a short adult flight is in-
creased. The second and third broods of 1942 are to be found indicated in the
figure a few weeks earlier than in the preceding year. As 1942 was a warmer year
for Mono Lake than was 1941, an earlier start in larval development in the spring,
with a consequent shift forward in the successive broods, would thus be expected.
The two 1940 samples at Mono Lake show no yellow butterflies present at all.
Therefore, it would appear that they were obtained in a yellow interbrood period
(Fig. 1).
The frequency at Round Valley does not follow this sequence of events
(Fig. 1). Neither the 1941 nor the 1942 samples show any correlation with those
%
80
70
60
50
40-
30
20 •
\ A ,
|Q. M0t*0 LAKE ------- l( ' / \'o
ROUND VALLEY - \ / \ \' ,''
\ / / \ V" /
o ...... - v — •«..-.
APRIL MAY JUNE JULY AUG. SEPT. OCT
FIGURE 1. Frequency of yellow to orange butterflies at Mono Lake and Round Valley,
California, throughout the season. Note the complete absence of yellow at certain times at
Mono Lake as compared with Round Valley.
of Mono Lake (Table I). This shows the complete lack of intermixing between
the two places though they are only fifty miles apart. The 1941 Round Valley
curve is high in late June (60 per cent yellow) and drops to a low in late July (25
per cent yellow). A rise occurs in mid-August (53 per cent), with a subsequent
drop again the first of September (34 per cent), and then a last rise in early October
(55 per cent). If these fluctuations represent successive broods not completely
separated one from the other, then there are many more generations present per
year at Round Valley than at Mono Lake. This would be expected considering
the warmer climate at the former place (Round Valley is at an elevation of 4500
feet and Mono Lake at 6500 feet). Latitudinal differences in brood number per
year parallel these altitudinal ones. There are three generations per year near
Washington, D. C. (and Mono Lake), two generations near Hanover, N. H. (and
46
WILLIAM HOVANITZ
central British Columbia) and one generation in Alaska and Yukon Territory.
At Round Valley there are probably four or more.
The 1942 samples are more extensive at Round Valley than are those of 1941
(Table I). There is a low of yellow (40 per cent) in early spring, rising to a high
within the month of 75 per cent and later 79 per cent, with a rather constant
frequency of 65 per cent yellow the remainder of the year. This curve shows
little evidence of a series of broods or generations during the year. At the rather
high temperatures prevailing in the valley during the summer (around 40° C.
TABLE I
The frequency of the yellow-race as compared with the orange-race butterflies at Mono Lake and Round
Valley, Calif. Standard errors are used in this and the other tables. The "many"
is not included in the figures of totals but indicates the presence of yellow alone.
Round Valley
Mono Lake
% yellow
N
% yellow
N
1940
—
Aug. 11
—
—
0.0
105
Oct. 20
—
—
0.0
46
1941
50.50 ± 2.89
299
5.84 ± 0.63
1,387
May 4
many
many
—
—
May 19
—
—
46.27
75
June 8
—
—
0.0
91
June 24
61.19
134
0.0
70
JulyS
50.82
61
0.89
678
July 26
22.92
48
12.66
237
Aug. 15
52.63
19
4.21
95
Sept. 2
33.33
15
0.0
20
Oct. 4
54.55
22
9.01
121
1942
65.96 ± 1.50
987
10.68 ± 0.88
1,236
April 1
40.82
49
—
—
April 25
74.55
110
—
—
June 12
77.14
140
0.0
many
July 7, 8
63.70
540
16.16
396
Aug. 6, 7
65.22
69
0.0
434
Sept. 16
65.82
79
10.67
406
1940-41-42
62.36 ± 1.35
1,286
7.68 ±0.51
2,774
during the day, and fluctuating but not very cool at night), the succession of
generations would be at about one month intervals. The samples were made at
this interval of time, so it is quite possible that the sampling periods coincided
with the periods of adult emergence. Were this the case, the results would show
a rather constant seasonal frequency. On the other hand, it is possible that the
variations in development rate between individuals owing to micro-temperature
differences in the locality have completely eliminated the inter-brood population
minima. This has been shown to be partially true for the second and third broods
in the vicinity of Washington, D. C., as well as for New York state. In these
HYBRIDIZATION IN BUTTERFLIES
47
places, only the breaks between broods one and two are clearly defined by the
absence of adults.
A higher frequency of yellow at Round Valley than at Mono Lake, at all
times, is apparent (Fig. 1). Several factors combine to create this difference:
(1) more larval food is present at Round Valley (Trifolium), (2) Round Valley
is farther ecologically from the source of the migrant orange-race individuals (San
Joaquin Valley), for these are more likely to stop in the mountain meadows than
to proceed through the desert to Round Valley. The frequency of yellow is given
as compared with orange. When the orange frequency goes down, the yellow
will appear to rise in the curve. (3) The longer and warmer growing season at
Round Valley gives more time for the resident population size to be built up.
This has been shown elsewhere by the increased numbers of individuals in the
second and third broods at Washington, D. C., and New York as compared with
the first spring brood.
TABLE II
The frequency of intermediates in the mixed population of orange and yellow races of Colias chryso-
theme at Round Valley, California. (The total given in Table I does not
include intermediates; hence, it is smaller than that given here.)
1941
1942
% intermediates
% intermediates
May 4
— .
many
April 1
10.91
55
June 24
14.65
157
April 25
7.56
119
July 5
11.59
69
June 12
14.15
163
July 26
9.43
53
July?, 8
6.08
575
Aug. 15
32.14
28
Aug. 6, 7
6.76
74
Sept. 2
0.0
15
Sept. 16
12.22
90
Oct. 4
0.0
22
1942
8.27 ±0.84
1,076
1941
13.08 ± 1.82
344
1941-42
9.44 ± 0.78
1,420
A higher frequency of yellows at Round Valley in 1942 as compared with the
1941 samples is also indicated. The latter samples were obtained in a mixed
alfalfa-red clover field at the periphery of the large meadow which constitutes the
primary ecologic niche for the yellow-race. The 1942 samples were made at a
different field one mile from the latter (containing alfalfa, red-clover, white clover
and native perennial clovers) in the center of the meadow. This field would be
in the midst of the population for the yellow-race whereas the former field is on
the periphery. For the migratory orange-race (Hovanitz, 1943d), no part of
the meadow would constitute a population center. The higher frequency of
yellow in 1942, therefore, can be accounted for by this change in position of the
place sampled.
Hybridization Between the Races
Genetic data on crosses involving the races and on progeny from wild inter-
mediates between the races indicate that crossing is easily possible and occurs
48 WILLIAM HOVANITZ
frequently (Hovanitz, 1943b). Also, the indications are that there is no genetic
sterility between the races. The FI is an exact intermediate of a light orange
color; F2 and backcrosses give the range of intermediates expected on a multiple
factor distribution of genes.
The range of colors from the parental types through the intermediates is
given in a range from yellow to orange of 1 to 10. From genetic results, it is
known that grades 1 and 2 are pure parental types, breeding true for the yellow
race. In the pure populations of orange race, there is a range of yellow to orange
from 1 to 10 but from about 1 to 7 or 8 these are exceedingly rare (Hovanitz,
1943e). Therefore, grades 8 to 10 in the males and 7 to 10 in the females are
considered as "parental types" for the Round Valley population. It is under-
stood that grades 7 or 8 may be intermediates or that some lower grades may be
parental types but that these will be insignificantly small.
40' ••.....
1941
1942
30-
20-
10-
APRIL MAY JUNE JULY AUG. SEPT. OCT.
FIGURE 2. Frequency of intermediates between yellow and orange in the population at
Round Valley, California, during the two years 1941-42.
On the basis of grades 3 through 7 in the males and 3 through 6 in the females,
the frequency of intermediates in the Round Valley populations have been calcu-
lated (Table II). It is seen that there is but little seasonal change in the abun-
dance of intermediates (Fig. 2). A high of 30 per cent in August 1941 is possibly
a result of the small sample size. An average of about 10 per cent intermediates
is usual.
Range of Wild Intermediates
The statistical consequences of continued interbreeding between the orange
and yellow races should be a single race combining the characteristics of each
parental type. But the two races have maintained their primary discreteness
after more than 70 years of such interbreeding, and probably for many centuries
(Hovanitz, 1943b; 1943c). Were the interbreeding only of very recent origin,
the hybrid range would show a very high frequency of FI intermediates (grades
5 or 6) and a lower range of F2, F3 and backcross intermediates (grades 3-4, 7-8).
The data on wild individuals (Fig. 3) do not show this higher frequency of FI to
HYBRIDIZATION IN BUTTERFLIES
49
any great extent. The female curve may be masked by the normally low orange
female grades. The male range shows a somewhat higher frequency of grade 5
than the other intermediates. The lack of the FI intermediates compared with
123456789 10
1234 56 7 8 9 10
FIGURE 3. Histograms showing the range of variation in the intermediates between the
orange and yellow races at Round Valley, California, 1941-42. Male on left and female on right.
The smaller numbers represent the numbers of individuals in a given class and the larger represent
the grade or class of intermediates.
Fn intermediates may be due to many factors of which a general lower viability
seems to be the most likely (Hovanitz, 1943b).
DISCUSSION
The data on the existence of the two races of Colias living in the same locality
suggest how ecologic and physiological differences can be maintained in units
50 WILLIAM HOVANITZ
which may be called species. The races are not here called species for some genes
are easily and often interexchanged (Hovanitz, 1943b). However, other genes
are not effectively segregated in this way. This suggests that the significant gene
complex characterizing each race and giving it individuality is not broken down
in hybrid crosses.
Since the color difference separating the races is a multiple factor one and these
factors are segregated independently of the basic complex, it might still be ex-
pected that a complete intermediate population would be produced, separated
only by the non-visible basic complex. The reason for this lack of complete
blending of characters probably lies in a combination of the following conditions:
(a) Sexual selection (Hovanitz, 1943b) may prevent sufficient intercrossing to
be effective.
(&) Eggs genetically determined to be yellow-complex laid on alfalfa will later
result in sterile adults or the subsequent larvae may die; also the reciprocal on
red clover (Hovanitz, 1943; 1943b).
(c) The intermediates of all types are probably less viable than the parental
types and many of them will be sterile on the food plant upon which they feed
(Hovanitz, 1942; 1943b).
(d~) The diapause associated with the one complex (Hovanitz, 1942, 1943b)
tends to keep the races ecologically separated.
(e) The supplementary color genes of each normal type probably act better
in unison with the basic complex than any intermediate segregation of genes.
(/) The different ecological niche occupied by the food plants necessary for
each complex aids in preventing hybridization (Hovanitz, 1943c).
Summary
1. Two localities where the two races of Colias chrysotheme occur together are
described (Mono Lake and Round Valley, Calif.).
2. In these places, the yellow-race has definite broods during the season. The
orange race apparently does not.
3. The yellow-race has more seasonal generations when a population is at a
lower elevation (Round Valley) than at a higher elevation (Mono Lake). This
compares with latitudinal differences of the same type.
4. The two localities are 50 miles apart, but show no correlation in seasonal
generations.
5. The yellow-race generations at the higher elevation are separated by inter-
brood periods with no adults. At the lower elevation, the generations merge one
into the other.
6. Hybrid intermediates are present at one locality rather constantly at a
frequency of about 10 per cent.
7. The range of color intermediates is not trimodal, but a U-shaped curve.
This is probably due to a low viability of the FI. A trimodal curve is expected
under conditions of very recent hybridization and all intermediates with long-
time hybridization.
8. Several reasons are given to account for the lack of complete blending be-
tween the races after years of hybridization.
HYBRIDIZATION IN BUTTERFLIES 51
A cknowledgments
This work was carried on through the encouragement of Professors T. H.
Morgan and A. H. Sturtevant to whom the author is grateful.
LITERATURE CITED
HOVANITZ, W., 1942. The biology of racial or species differences in Colias, Bull. Ecol. Soc. Amer.,
23: 68 (Abstract).
HOVANITZ, W., 1943a. The nomenclature of the Colias chrysotheme complex in North America.
American Museum Novitates (in press).
HOVANITZ, W., 1943b. Genetic data on the two races of Colias chrysotheme in North America
and on a white form occurring in each. (Awaking publication.)
HOVANITZ, W., 1943c. The ecological significance of the color phases of Colias chrysotheme in
North America. Ecology (in press).
HOVANITZ, W., 1943d. The distribution of gene frequencies in wild populations of Colias. Genetics
(in press).
HOVANITZ, W., 1943e. The pattern elements of the North American Colias of the chrysotheme
group. (Awaiting publication.)
SPECIES DIFFERENCES IN RATES OF OSMOTIC HEMOLYSIS
WITHIN THE GENUS PEROMYSCUS *
HARRY P. LEVINE
(Department of Zoology, University of Vermont, Burlington)
INTRODUCTION
That definite species differences exist in the properties of the red cell membrane
has been recognized at least since the studies of Rywosch in 1907. The possible
significance of such specific differences in regard to zoological classification and
animal identification has been pointed out by Jacobs (1931). Investigation of
the rates of osmotic hemolysis in approximately 50 species of vertebrates led to
the conclusion that "not only may individual species be identified but frequently
unmistakable evidences of zoological relationship may be traced throughout a
group of similar forms." In 1938 Jacobs and collaborators demonstrated striking
differences in the permeability properties of the erythrocytes of the rat and mouse
representing closely related genera. The purpose of the present investigation
was to demonstrate measurable and consistent differences in the rates of hemolysis
among a number of species within the genus Peromyscus.
MATERIALS AND METHODS
The mice used in this investigation consisted of four species representing
different degrees of taxonomic relationship (Miller, 1923) from widely separated
geographical regions as follows:
Subgenus Haplomylomys Osgood
P. eremicus fraterculus — La Jolla, California
Subgenus Peromyscus Gloger
Species group — leucopus
P. leucopus noveboracensis — Vermont; Moville, Iowa
P. gossypinus palmarius — Sebring, Florida
Species group — truei
P. truei truei — Deadman Flat, Arizona
In addition, the guinea pig (Cavia cobaya) representing a distantly related
rodent species was used for purposes of contrast.
Blood was obtained from each mouse under light ether anesthesia by cardiac
puncture after the method of Hicks and Little (1931). About 0.5 cc. could be
removed from a mouse without fatality. The blood was immediately expressed
into a small beaker containing about 10 cc. of 0.9 per cent saline and defibrinated
by stirring. The suspension was then washed down by centrifuge and the cells
restored to the original blood volume with saline.
* Preliminary report presented at the 24th annual meeting of the American Society of Mam-
malogists in New York City, April 2, 1942.
52
SPECIES DIFFERENCES IN HEMOLYSIS RATES
53
The substances employed in the hemolysis studies were 0.3 molar solutions
in distilled water of non-electrolytes including ethylene glycol, glycerol and ery-
thritol representing progressively larger polyhydric alcohol molecules, and
thiourea.
The method of determining rates of hemolysis was essentially that described
by Jacobs (1930). To 5 cc. of one of the above solutions in a test tube in a water
bath maintained at 20° C. was quickly added one drop of blood on a specially
prepared plunger which simultaneously stirred the cells, producing an even
suspension. With the aid of a stop-watch the time for 75 per cent hemolysis of
the cells was determined by comparison with a standard suspension (one drop of
the same blood in 20 cc. of saline) in a test tube adjacent to that containing the
hemolysing suspension. This comparison was effected by means of a thin band
of light viewed through the test tubes. Approximately 75 per cent hemolysis
was attained when the band of light was visible in the hemolysing suspension to
the same degree as in the standard. In practice the blood to be tested was so
TABLE I
Species differences in rates of osmotic hemolysis
Time in seconds for 75 per cent hemolysis at 20° C. in 0.3M
Ethylene glycol
Glycerol
Erythritol
Thiourea
Species
No.
Low
High
Ave.
Low
High
Ave.
Low
High
Ave.
Low
High
Ave.
P. lencopus
17
4.7
6.4
5.6
7.0
12.3
9.5
20.6
49.0
31.8
10.7
15.3
13.3
P. gossypinus
14
5.6
7.8
7.1
15.0
28.5
22.1
47.0
195.0
110.0
13.3
21.7
19.4
P. truei
6
6.8
8.0
7.3
33.6
44.3
39.2
150.0
250.0
193.0
28.7
36.0
32.1
P. eremicus
15
5.6
6.7
6.0
31.0
58.5
44.3
150.0
465.0
259.0
16.8
28.5
23.3
Cavia cobaya
3
10.6
15.4
13.6
130.0
223.0
178.0
>30 hrs. <42 hrs.
116.0
143.0
126.0
adjusted with saline that the band of light was just barely visible through the
standard suspension since this offered the most easily recognized end point.
In performing the experiments test tubes were carefully chosen for uniformity,
standard suspensions were prepared as soon as the blood samples were obtained,
and hemolysis rates were determined immediately. All tests were performed in
duplicate whenever possible. Remaining portions of blood samples were kept in
refrigeration storage at approximately 4° C. Except for certain storage experi-
ments where pooled blood was used, hemolysis rates were obtained with eryth-
rocytes from individual animals.
EXPERIMENTAL RESULTS
The method of determining rates of hemolysis as described above was very
simple and apparently crude, but with proper care the results of tests performed
in duplicate proved to be markedly consistent. Variation in duplicate measure-
ments of the time for 75 per cent hemolysis of the red cells in any one of the
solutions rarely exceeded 10 per cent and most often was less than 5 per cent.
With practice, especially in preparing suitable standard suspensions, duplication
54
HARRY P. LEVINE
was brought to within 2 per cent. It was reasonable to assume, therefore, that
the differences in hemolysis times obtained here between one species and another
represented true specific differences.
The times for 75 per cent hemolysis of the erythrocytes of the species investi-
gated are summarized in Table I. Evidence of zoological relationship is readily
apparent. When compared with the rate of hemolysis of guinea pig (Cavia)
erythrocytes, the hemolysis rates of all the Peromyscus erythrocytes appear to
be of the same order of magnitude. With erythritol, for example, the difference
TABLE II
Comparison of glycerol and thiourea times and G/T ratios of four species in the genus Peromycus
(temperature 20° C.)
hemolysis time in glycerol
G/T ratio =
hemolysis time in thiourea
Time in seconds for 75 per cent hemolysis
G/T Ratio
0.3M Glycerol
0.3M Thiourea
P. leucopus
12.2
13.8
0.88
8.1
13.7
0.59
12.3
15.3
0.80
7.0
10.7
0.65
Ave. 9.5
13.3
0.71
P. gossypinus
28.5
21.4
1.33
22.5
20.8
1.08
24.4
21.7
1.12
15.0
13.3
1.13
Ave. 22.1
19.4
1.14
P. truei
44.3
33.2
1.33
33.6
29.8
1.13
43.4
36.0
1.21
35.6
28.7
1.24
Ave. 39.2
32.1
1.22
P. eremicus
58.5
27.3
2.11
40.2
22.9
1.76
54.6
27.5
1.98
31.0
16.8
1.85
Ave. 44.4
23.3
1.90
in hemolysis time between leucopus cells and eremicus cells (of the order 1 : 8)
is small when compared with the difference between eremicus cells and guinea
pig cells (1 : 540). On the other hand, consistent differences in hemolysis rates
among the species within the genus are demonstrable. Leucopus cells are most
readily hemolysed by each of the permeating substances; gossypinus cells are
hemolysed at a somewhat slower rate. Generally truei and eremicus cells are
hemolysed less rapidly than either leucopus or gossypinus cells. It is interesting
to note in this regard that leucopus and gossypinus are placed taxonomically
within the same species group.
SPECIES DIFFERENCES IN HEMOLYSIS RATES
55
The rates of osmotic hemolysis in glycerol especially often reveal striking
specific differences and sometimes offer evidence of relationship (Jacobs, 1931;
1938). From Table I it can be seen that all the Peromyscus red cells attain the
condition of 75 per cent hemolysis in less than one minute. Yet the hemolysis
times for the red cells of each species are apparently confined to definite limits
within this time.
According to Jacobs and associates (1938), comparison of the rates of osmotic
hemolysis in glycerol and thiourea within a species may provide an index for
species identification. Table II records in the first two columns the hemolysis
times in glycerol and in thiourea respectively for each species of mouse investi-
gated. The figures in the third column (G/T ratio) are obtained by dividing the
glycerol hemolysis time by the thiourea hemolysis time. The data have been
selected to show the extent of variation found in each species. The average
figure for each species is the arithmetic mean of the results for all individuals
o
<
20 30
TIME IN SECONDS
40
5O
60
FIGURE 1. Species differentiation by osmotic hemolysis. Each dot represents an individual
plotted along the abscissa in terms of the time for 75 per cent hemolysis in 0.3 molar glycerol at
20° C. and along the ordinate in terms of the G/T ratio. The hollow rectangles represent different
species:
A = P. leucopus
D = P. eremicus
B = P. gossypinus
C = P. truei
studied within each species. It is evident from the table that the glycerol/
thiourea ratio is constant for each species within fairly narrow limits. Leucopus
which has the lowest ratio and eremicus which has the highest ratio are readily
separated from gossypinus and truei. Although the latter two species exhibit
similar ratios, examination of the first two columns in Table II reveals that in
the absolute times for hemolysis in glycerol and in thiourea they are readily
differentiated.
Figure 1 records graphically the results which have been summarized in Table
II. Each mouse investigated in the present study has been plotted with regard
to erythrocyte hemolysis in glycerol (along the abscissa) and with regard to the
glycerol/thiourea ratio (along the ordinate). The hollow rectangles enclose all
the individuals within a species. This figure shows in a striking way that it may
be possible to determine the species to which an individual belongs by the ap-
propriate hemolysis tests. For example, at one stage in the course of these
56 HARRY P. LEVINE
experiments a colleague kindly provided two blood samples without revealing
the species from which they had been obtained. Hemolysis tests provided the
following results:
Time in seconds for 75 per cent hemolysis at 20° C.
0.3M glycerol
0.3M thiourea
G/T ratio
Mouse No. 1
Mouse No. 2
12.1
8.5
15.0
12.8
0.80
0.67
Both mice were correctly identified as leucopus.
Some evidence of zoological relationship is apparent in the glycerol/thiourea
ratios obtained in this study. As can be noted in Table II, the ratios for leu-
copus, gossypinus and truei which are placed in the same taxonomic subgenus are
all near one as a constant, while the ratio for eremicus which is placed in another
subgenus is near two.
At the inception of this investigation some disconcerting variations in he-
molysis times occurred within each species of Peromyscus. This led to an in-
vestigation of the effect of storage upon the rate of hemolysis of the red cells.
In order to obtain a sufficient quantity for this purpose, it was necessary to use
pooled blood of each Peromyscus species, whereas blood from individual guinea
pigs was employed. Otherwise all blood samples were treated identically. Fig-
ure 2 shows the typical effect of storage upon the hemolysis rates of the Peromyscus
and guinea pig red cells. Days in storage are plotted against the hemolysis time
in glycerol. The red cells of eachTof the species within the genus Peromyscus
show a marked and continued increase in hemolysis time upon storage while the
red cells of the guinea pig show very little change during the same period of storage.
The reason for this interesting storage effect has not yet been determined.
DISCUSSION
Physiological and biochemical studies of blood have produced results both of
broad evolutionary interest and also of value in the field of animal classification
and identification. The evolutionary significance of results obtained from the
studies of the osmotic pressures of blood (Scott, 1916) is well recognized. The
extensive work of Reichert and Brown (1909) on the crystallography of hemo-
globin among different species has provided convincing evidence of biochemical
relationships among animals in general accord with the accepted taxonomic classi-
fication. The versatile and rapidly expanding field of systematic serology (see
Boyden, 1942) has been employed on the one hand in the study of the possible
origin of vertebrates (Wilhelmi, 1942), and on the other hand, in the investigation
of the genetic basis for biochemical differences in the serum and blood cells of
species and species-hybrids (Irwin and Cole, 1936; Irwin and Cumley, 1942).
The present investigation has revealed that consistent and measurable differ-
ences in the rates of hemolysis of the erythrocytes among very closely related
species can be employed successfully to differentiate one species from another.
Especially with regard to glycerol penetration, confirming observations by Jacobs,
SPECIES DIFFERENCES IN HEMOLYSIS RATES
57
and with regard to the glycerol/thiourea ratio the results indicate zoological rela-
tionship in general agreement with the existing system of classification. Whether
such agreement between morphological classification and rate of osmotic he-
molysis will always hold among closely related species can be determined only
by further investigation.
170
160
iP LEUCOPUS
• P GOSSYPINUS
»P EREMICUS
CAVIA COBAYA
DAYS I N STORAGE
FIGURE 2. The effect of storage upon the rate of osmotic hemolysis (75 per cent) in 0.3
molar glycerol at 20° C. (Blood cells stored in 0.9 per cent NaCl at approximately 4° C.)
Preliminary studies on four offspring of a species-cross between leucopus and
gossypinus indicate that these differences may be subject to genetic analysis
although as yet the data are not sufficient for definite conclusions. Table III
shows that in their hemolysis times in different substances the hybrid red cells
are very similar to those of the leucopus parent stock while the values for the
glycerol/thiourea ratio lie between those of the two parent stocks.
Specific differences in the properties of the cell membrane have introduced a
58
HARRY P. LEVINE
complicating feature to the problem of cell permeability, yet an understanding
of the nature of such specific differences may go far towards a better under-
standing of the factors determining the permeability of the cell membrane in
general. In the meantime collection of further data on species differences in
erythrocyte permeability will serve the useful purpose of developing a physiological
means of animal identification.
The author is deeply indebted to Dr. Paul A. Moody who gave unstintedly
of his mice and of his time when requested ; to Dr. Lee R. Dice of the University
of Michigan who provided some of the mice from which the present stock was
originated; and especially to Dr. Merkel H. Jacobs of the University of Pennsyl-
vania, under whose guidance the author became acquainted with the described
hemolysis techniques at the Marine Biological Laboratory, at Woods Hole,
Massachusetts. The author is further indebted to Dr. Jacobs for his kindness
in reading the manuscript and for his valuable suggestions.
TABLE III
Comparison of hemolysis times and G/T ratios of a species hybrid and its parent stocks
Species
Time in seconds for 75 per cent hemolysis at 20° C. in 0.3M
G/T ratio
Ethylene glycol
Glycerol
Erythritol
Thiourea
*P. leucopus noveboracensis
*P. gossypinns pal mar ins
leucopus-gossypinits hybrids
5.6
7.1
4.8
5.1
9.5
22.1
8.0
10.4
31.8
110.0
28.0
39.0
13.3
19.4
8.6
10.1
0.71
1.14
0.93
0.97
5.2
10.2
34.0
10.5
1.03
4.9
7.8
20.0
8.7
1.12
Average of the species.
SUMMARY
The erythrocytes of four species of mice within the genus Peromyscus were
studied with regard to their rates of osmotic hemolysis in ethylene glycol, glycerol,
erythritol and thiourea. Consistent species differences in hemolysis times were
demonstrated by which it was possible in the case of the individuals studied to
identify each species with certainty. Evidence of zoological relationship was
apparent in the results.
Refrigeration storage of Peromyscus erythrocytes resulted in progressively
decreased rates of hemolysis. Storage of Cavia (guinea pig) erythrocytes had
very little effect upon their rates of hemolysis.
LITERATURE CITED
BOYDEN, A., 1942. Systematic serology: A critical appreciation. Physiol. Zool., 15: 109-145.
HICKS, R. A., AND C. C. LITTLE, 1931. The blood relationships of four strains of mice. Genetics,
16: 397-421.
IRWIN, M. R., AND L. J. COLE, 1936. Immunogenetic studies of species and species hybrids in
doves, and the separation of species — specific substances in the backcross. Jour. Exp.
Zool., 73: 85-108.
SPECIES DIFFERENCES IN HEMOLYSIS RATES 59
IRWIN, M. R., AND R. \V. CUMLEY, 1942. Immunogenetic studies of species; qualitative differ-
ences in the serum of backcross progeny following a generic cross in birds. Genetics,
27: 228-237.
JACOBS, M. H., 1930. Osmotic properties of the erythrocyte. I. A simple method for studying
the rate of hemolysis. Biol. Bull, 58: 104-122.
JACOBS, M. H., 1931. Osmotic hemolysis and zoological classification. Proc. Amer. Phil. Soc.,
70: 363-370.
JACOBS, M. H., H. N. CLASSMAN AND A. K. PARPART, 1938. Osmotic properties of the erythro-
cyte. IX. Differences in the permeability of the erythrocytes of two closely related
species. Jour. Cell, and Comp. Physiol., 11: 479-494.
MILLER, G. S., JR., 1923. List of North American recent mammals. U. S. Nat. Mus. Bull. 128.
REICHERT, E. T., AND A. P. BROWN, 1909. The crystallography of hemoglobins. Carnegie Inst.
of Wash. Pub. No. 116.
RYWOSCH, O., 1907. Vergleichende Untersuchungen iiber die Resistenz der Erythrocyten einiger
Saugethiere gegen hamolytische Agentien. Pfliiger Archiv., 116: 229-251.
SCOTT, G. G., 1916. The evolutionary significance of the osmotic pressure of the blood. Amer.
Nat., 50: 641-663.
WlLHELMl, R. W., 1942. The application of the precipitin technique to theories concerning the
origin of the vertebrates. Biol. Bull., 82: 179-189.
GERMARIAL DIFFERENCES AND THE PRODUCTION
OF APHID TYPES*
CHESTER A. LAWSON
(Department of Zoology, Michigan State College, East Lansing, Michigan)
INTRODUCTION
If germaria exercise any control over the development of differential characters
in female aphids (Lawson, 1939; 1940) it is possible that they would give evidence
of this control by exhibiting structural peculiarities correlated with the production
of specific aphid types. To investigate this possibility the germaria of partheno-
genetic females producing different aphid types were compared.
THE GERMARIA
Each adult germarium contains two types of cells, nurse cells and germ cells.
The nurse cells are larger than the germ cells and make up the bulk of the ger-
marium, so if the germarium controls development it is possible that this control
stems from the nurse cells. Their prominence in the germarium at least gives
them first choice of the parts to be tested, so in this study the nurse cells only are
compared.
The nurse cells of all parthenogenetic germaria are essentially alike (Figs. 1,
2, 3, 4). Each nurse cell is roughly pyramidal in shape (triangular in section)
with the base at the periphery of the germarium and the apex in the center. The
nucleus lies near the base of the pyramid and is covered on its outer edge and sides
by a thin layer of cytoplasm. On the inner border of the nucleus the cytoplasm
is thicker and extends inward toward the center of the germarium forming the
apex of the pyramid. The cytoplasm seldom forms a sharp point in the center,
for here it blends with the secreted substance found in the center of all germaria.
The exact line of demarcation between cytoplasm and secreted material is difficult
to see. The nuclei of all nurse cells are relatively large and each contains a large
elliptical nuceolus and chromatin in the form of thin rods or prophase strands that
are interconnected by a fine threadlike network.
In comparing the germaria of different aphid types, structural differences were
sought that would serve to differentiate among them. Of several possible struc-
tural differences only one stands out with any consistency. This is a size differ-
ence. To test the reality of this apparent difference measurements were made
and compared of the entire germarium and of individual nuclei within the
nurse cells.
* Thanks are due to Professor W. D. Baten of the Mathematics Department who assisted
with the calculations and to Professor C. P. Swanson of the Botany Department who made the
photomicrographs. Part of this work was done at the Franz Theodore Stone Laboratory, Put-
In-Bay, Ohio.
60
PRODUCTION OF APHID TYPES
61
•
r/
'
• *
1
• *
* ** y
v«
•
^
• V,
•. •
0>
FIGURES 1-5. Cross-sections of adult aphid germaria. Figure 1. Winged parthenogenetic
female producing gamic embryos (1455 X). Figure 2. Winged parthenogenetic female producing
parthenogenetic female embryos (1455 X). Figure 3. Wingless parthenogenetic female produc-
ing parthenogenetic female embryos (1455 X). Figure 4. Wingless parthenogenetic female
producing male embryos (1455 X). Figure 5. Adult^gamic female (675 X).
62
CHESTER A. LAWSON
As each germarium is approximately spherical in shape its center cross section
is circular or elliptical. The diameters of this cross-section were measured in
micra and the area computed and this figure used to represent the size of the
germarium. A better method of comparing the germaria would be to compare
volumes. In order to calculate the volume of any one germarium it is necessary
to have three diameters because very few of the germaria are perfect spheres.
Two of these are easily measured on the center cross section. The third can be
gotten by counting the number of cross sections of the germarium. However, no
great reliance can be placed on a measurement arrived at in this manner. Each
cross section is ten micra in thickness except the first and the last. These two
vary from a fraction of one to ten micra, and as the actual thickness cannot be
determined the third diameter has a possible error of twenty micra. Because of
this error no confidence can be placed in the calculated volumes and it seems best
to restrict the comparisons to the more accurately measureable center areas of
the germaria. An occasional irregularity in the circumference of the cross sec-
tions introduces a source of error which is probably not great enough to dis-
count major size differences, but may affect the results in comparison of minor
differences.
Each aphid has nine or ten germaria and all of these that could be measured
accurately were measured and all measurements from one type of aphid were
grouped and treated statistical ly.
The means and standard deviations of the area of the center cross section of
the adult germaria are given in Table I. The germaria of the male-producing
rr\ T
FABLE I
.1 comparison of the areas in square micra of germarial center section
Type of female
Contained embryos
n
Mean
Standard
deviation
1.
wingless parth.
males
103
1474±42
422±29
2.
wingless parth.
parth. females
172
731 ±14
190±10
3.
\\inged parth.
parth. females
126
599±16
178±11
4.
winged parth.
gamic females
127
567±10
115±7
wingless parthenogenetic females (Fig. 4) are larger than those of the wingless
females producing parthenogenetic females (Fig. 3) and these in turn are larger
than the germaria of winged females (Figs. 1 and 3). The differences between
the means are statistically significant for all except the two winged types.
A difference between two means is considered significant when it is at least
twice the standard error of the difference between means.
The Nurse Cell Nuclei
The nuclei of the nurse cells also were measured and compared. All the nuclei
in any one germarium were not measured, but only those that were spherical.
Many of the nuclei formed long ellipses or varied from the spherical unevenly.
These nuclei were rejected in order to reduce the error of measurement and also
to reduce the labor. If spherical nuclei only are used, one measurement, the
PRODUCTION OF APHID TYPES
63
diameter, is sufficient; and from this the volume can be calculated. This selection
may introduce an error in the results if the shape of the fixed nucleus is correlated
with its size, which is unlikely; or if an insufficient number of nuclei are measured
in any one aphid type. It is believed that the number measured is sufficiently
large to evade this source of error.
The means and standard deviations of nurse cell nuclear volumes in cubic
micra are given in Tables II and III.
TABLK II
-1 comparison of nurse cell nuclear volume measured in cubic micra
Type of female
Embryos
n
Mean
Standard
deviation
1.
gamic
100
5180±314
3140±222
2.
wingless parth.
males
205
953±28
402 ±20
3.
wingless parth.
parth. females
408
326±7
140±4
4.
winged parth.
parth. females
351
283 ±7
130±5
5.
winged parth.
gamic females
200
151±5
64±3
TABLE III
Means and standard deviations in cubic micra of nurse cell nuclear volume of parthenogenetic females
producing different types of parthenogenetic embryos
Type of aphid
Embryos
n
Mean
Standard
deviation
1 . wingless
winged and wingless
200
302 ±9
125±6
2. wingless
winged
208
348±10
148 ±7
3. winged
winged and wingle^
351
283±7
130±5
4. winged
winged
198
279±8
129±6
5. winged
wingless
134
301±11
130±8
In Table II are listed the means and standard deviations of the five major
aphid types. The differences in the mean nuclear volumes are all statistically
significant. Thus the nurse cell nuclei of gamic female germaria (Fig. 5) are
larger than any of the others, those of wingless females producing males (Fig. 4)
are smaller than the gamic nuclei, but larger than any other parthenogenetic
nurse cell nuclei. The wingless females producing parthenogenetic females (Fig.
3) have nurse cell nuclei that are smaller than the male-producing type but larger
than those in winged females, while the winged female nurse cell nuclei are smaller
than any of the others. There is also a nuclear size difference between the two
types of winged females. The winged females producing gamic females (Fig. 1)
have smaller nuclei than those producing parthenogenetic females (Fig. 2). In
comparing Figures 1 to 5 it should be noted that the magnification of Figure 5
is approximately one-half that of Figures 1, 2, 3, 4.
The size differences shown by the nuclear measurements are in the same direc-
tion as those shown by the germarial measurements which suggest that the size of
the entire germarium is clue to the size of the nurse cells. One exception to this
is seen in the two sets of measurements of the winged parthenogenetic females.
64 CHESTER A. LAWSON
In comparing measurements of germarial center areas (Table I) the winged females
producing parthenogenetic females and those producing gamic females are not
significantly different. The means are different and direction of difference is the
same as that of the nuclear size differences, but the difference is not statistically
significant. In comparing the nuclear measurements of these same winged
female types (Table II) a very large and significant difference appears.
One of the possible explanations is that no correlation exists between nuclear
size and germarial size but rather between germarial size and nuclear (cell)
number. If this is true the germaria of winged females producing gamic females
should have almost twice as many nuclei as the germaria of winged females pro-
ducing parthenogenetic females. A count revealed the same number in both
(average 20 to 22). Another possibility is that there might be twice as much cyto-
plasm in each nurse cell within the gamic producing germaria, or that the material
secreted by the nurse cells is excessive. These possible differences are not ap-
parent on comparing the two types of germaria (Figs. 1 and 2) hence it is likely
that there is some other explanation at present unknown. Also there remains the
possibility that a difference may exist between the germarial areas of the two
types of winged females (Table I) that is not shown in these calculations. The
number of individuals used for computing the means of the germarial areas are
one-half as many as are used in computing the means for nuclear volume of the
same individuals (Table II). If n were doubled for the germarial areas a sig-
nificant difference might appear.
In Table III are listed the means and standard deviations of parthenogenetic
females that arc producing parthenogenetic female offspring. The winged and
wingless adults are classified according to whether they are producing either
winged or wingless parthenogenetic female offspring or both.
The means are all about the same and none of the differences is statistically
significant except for number 2 (wingless females producing winged embryos).
This mean is significantly different from all in the table except number 5 (winged
females producing wingless offspring). Thus except for one case no size difference
is correlated with the production of parthenogenetic types and in this one case
the difference is not great so it is possible that some factor other than type of
offspring produced the difference.
If this interpretation is correct and there is no real size difference among the
nurse cell nuclei in Table III a change must be made in the interpretation of
Table II. In this table the mean nuclear sizes of number 3 (wingless partheno-
genetic females producing parthenogenetic female embryos) and number 4 (winged
parthenogenetic females producing parthenogenetic female embryos) are sig-
nificantly different. However, the calculation of the mean of 326 ± 7 of number
3 of Table II includes the data under number 2 of Table III. If these data are
eliminated from the calculations the mean becomes 302 ± 9 (a = 125 ± 6) and
the difference disappears between this mean and that of the number 4, Table II
(winged parthenogenetic females producing parthenogenetic female embryos).
Thus the group of data in Table III that shows a questionable difference causes
the difference between the winged and wingless parthenogenetic-producing females
in Table II. Hence 3 and 4 in Table II probably are not different. There re-
main, however, the differences among the other types which are so large that their
reality seems beyond doubt.
PRODUCTION OF APHID TYPES 65
Germaria and Embryos of Winged-wingless Intermediates
A study of winged-wingless intermediates offers further evidence that the
nurse cell nuclear volume is correlated with the type of offspring produced. In
Table IV is presented a comparison of the mean nuclear volume of germarial
TABLE IV
A comparison of volumes in cubic micra of nurse cell nuclei in gerniaria of winged -wingless
parthenogenetic female intermediates with the type of embryos contained
in the vitellaria to which these gerniaria are attached
Mean nuclear volumes Types of embryos
1. 57±9 Gamic female
2. 63 ±9 Gamic female
3. 132±10 Parthenogenetic female
4. 140±9 Parthenogenetic female
5. 157 ±8 Gamic female
6. 194±11 Parthenogenetic female
7. 235 ±14 Gamic and parthenogenetic female
8. 272 ±15 Parthenogenetic female
9. 353 ±31 Parthenogenetic female and male
10. 381 ±37 Parthenogenetic female and male
11. 383 ±23 Parthenogenetic female and male
12. 383±23 Parthenogenetic female
13. 486±58 Parthenogenetic female and male
14. 559±61 Parthenogenetic female and male
15. 732±93 Male
16. 804 ±73 Male and gamic egg
17. 930 ±82 Male
nurse cells in individual winged-wingless intermediates and the type of embryos
contained within the ovarioles of the intermediates. From this comparison it is
evident that the intermediates having the smallest nuclear volume contain gamic
female embryos within their ovarioles, and that as the nuclear volume becomes
greater the embryos become parthenogenetic, then both parthenogenetic and male
(in which the older embryos are parthenogenetic) then all male embryos and finally
the intermediates having the largest nurse cell nuclear volume contain both male
embryos and gamic eggs.
This correlation is not exact for intermediates 3 and 4, Table IV, contain
parthenogenetic embryos while intermediate 5 has gamic embryos and also has a
larger mean nuclear volume than either 3 or 4. Also intermediates 11 and 12
have the same nuclear volume, even though number 11 has both male and par-
thenogenetic female embryos, while number 12 has parthenogenetic embryos only.
This irregularity may be due to the fact that all of the nuclei in any one inter-
mediate could not be measured accurately, or it may be due to the effect of some
unknown factor. In any case it seems reasonable to conclude that in winged-
wingless intermediates the size of the nurse cell nuclei is correlated in general
with the production of specific aphid types.
In one intermediate (17) there is an unusual germarium (Fig. 6) in which the
nuclei are of two distinct sizes. The germarium is partly divided in half; one-half
containing large nuclei (M = 2264 ± 390) the other half containing small nuclei
(M = 445 ± 53). The appearance of two distinct sizes of nuclei within one
germarium suggests that the size of any one nurse cell nucleus is determined by
66
CHESTER A. LAWSON
some factor within the germarium and perhaps within the individual nucleus
itself. What this factor might be is entirely hypothetical; however, the nuclear
size variation suggests polyploidy. No chromosome counts have been made as
yet, but as the nuclei of gamic female germaria are filled with small rod-shaped
chromosomes and are so much larger than any of the other types of nuclei it is
probable that there is more chromatin in the gamic nuclei than in the others.
* *
FIGURES 6-7. Figure 6. Abnormal germarium of a winged-wingless intermediate showing
nuclei of two sizes (675 X). Figure 7. Degenerate body (embryo?) found in ovariole of wingless
parthenogenetic female producing males (675 X).
PRODUCTION OF APHID TYPES 67
Are all intermediates physiologically wingless?
Shull (1940) has suggested that adult winged-wingless intermediates are not
physiologically intermediate but, rather, that they are wingless having progressed
during development from a winged to a wingless condition. The structural
characters become fixed in an intermediate condition during the transition and
remain so during the life of the aphid, but the physiological nature of the individual
continues changing until it is completely wingless. As a typical winged individual
produces gamic females during the gamic phase of the cycle while a wingless female
produces males, the physiological nature of the intermediates was determined by
examining the type of offspring produced by them. Thus, if an intermediate
produced males it was judged to be physiologically wingless. If it produced
gamic females it was winged. Shull concluded that all winged-wingless inter-
mediates are physiologically wingless.
An opposite conclusion is indicated by the evidence derived from the inter-
mediates used in this study. These intermediates produced both male and gamic
female embryos. Consequently some of them were physiologically winged and
some wingless.
Degeneration in male-producing wingless females
Wingless females that are producing males not only have distinctive germaria
but they also have degenerating cell masses within their ovarioles. The cell
masses (Fig. 7) occur in the ovarioles at any position though they were observed
most frequently at the end nearest the germarium. They are elliptical in longi-
tudinal section and circular in cross section. A vacuolated center area is usually
surrounded with a rim of densely staining pycnotic cells. What the degenerating
bodies are is questionable but their elliptical shape is similar to young embryos,
and furthermore, the rim of cells surrounding a vacuolated non-cellular center
area is typical of young male blastulae. Therefore, it is tentatively concluded that
the degenerating bodies are embryos that failed to continue development and are
being resorbecl. Why degenerating embryos should be characteristic of male-
producing wingless females remains an open question.
CONCLUSION
A correlation between the size of the germaria and their nurse cell nuclei and
the type of embryos produced seems established. Whether the germaria actually
control production of aphid types is still unknown.
SUMMARY
The areas of the center cross-section of adult germaria of parthenogenetic
female aphids producing different types of offspring were measured and compared.
From this comparison it is evident that the center cross-sections of the germaria
of male-producing wingless parthenogenetic females are larger than those of wing-
less females producing parthenogenetic females, and these in turn are larger than
the cross-section of winged female germaria. All winged females have the same
cross-sectional area whether they are producing parthenogenetic or gamic females.
68 CHESTER A. LAWSON
A comparison of the volume of the nurse cell nuclei shows that the nuclei of
gamic female germaria are larger than any of the others; those of wingless females
producing males are smaller than the gamic nuclei, but larger than any other
parthenogenetic nurse cell nuclei. The wingless and winged females producing
parthenogenetic females have nurse cell nuclei of the same size, while the nurse
cell nuclei of winged females producing gamic females are the smallest of all.
A correlation of the nurse cell nuclear volume of winged-wingless intermediates
with the embryos contained in the ovarioles supports the thesis that size of nuclei
and type of young produced are interdependent. Those intermediates that con-
tained gamic embryos have the smallest nuclei; those with the next in nuclear
size have both parthenogenetic and male embryos. The largest contain males
only or males and gamic eggs.
LITERATURE CITED
LAWSON, C. A., 1939. The significance of germaria in differentiation of ovarioles of female aphids.
Biol. Bull., 77: 135-145.
LAWSON, C. A., 1940. The developmental history of germaria in parthenogenetic female aphids.
Ohio. Jour. Sci., 40: 74-81.
SHULL, A. F., 1940. Adult intermediate-winged aphids not physiologically intermediate. Genet-
ics, 25: 287-298.
POLYDORA IN OYSTERS SUSPENDED IN THE WATER
VICTOR L. LOOSANOFF AND JAMES B. EXGLE
(Fish and Wildlife Service, Fishery Biological Laboratory, Milford, Connecticut)
INTRODUCTION
Among the numerous enemies of oysters the small Polychaete worms of the
genus Polydora have long been considered as very destructive. It has been re-
ported that sometimes these worms may be responsible for the complete dis-
appearance of extensive oyster beds. Such depredations were described by
Whitelegge (1890) and Roughley (1922, 1925) who were working in Australian
waters, where Polydora caused a heavy mortality among the native oysters. Both
authors identified the worm as P. ciliata. It is possible, however, that Whitelegge
was mistaken in his identification of the species. According to Wilson (1928)
"Whitelegge found the ova and larvae of a species of Polydora attached alongside
the adults to the walls of their burrows in oyster shells at Newcastle, in New
South Wales. He believed the species to be Polydora ciliata Johns, but his figure
of the egg-sacs resembles more closely that given by Soderstrom for Polydora ligni
Webster." If Whitelegge was actually mistaken then the destruction of the
oysters in Australian waters should be attributed to at least two species of
Polydora, namely, P. ciliata and P. ligni.
Several species of Polydora are common along our Atlantic Coast. Lunz
(1940, 1941) found that approximately 40 per cent of the oysters of South Caro-
lina waters are infested with P. ciliata. This author states in his latest paper that
he now has evidence or reports of infestation throughout the entire range of
distribution of the American oyster, 0. virginica, in North America. Nelson and
Stauber (1940) stated in a brief abstract that many oysters of New Jersey harbored
P. ligni Webster. This appears to be the same species which, in the opinion of
Wilson, Whitelegge was dealing with in Australia. Kavanagh (1940) found that
the Japanese oyster, 0. gigas, planted in Louisiana waters became infested with
P. ciliata. Takahashi (1937) reported that P. pacifica was quite commonly
present in the shells of the pearl oyster, Pinctada margaritifera.
Polydora or, as it is usually called, mud worm, is also known to infest shells
of mollusks other than oysters. Lebour (1907) found that the mussels of the
Northumberland beds of England were heavily infested with P. ciliata, and Field
(1922) stated that the same species occurs in shells of the mussel, M. edulis, living
in American waters.
Polydora usually gains entrance into the oyster while the worm is still in the
larval stage, or when very young (W7ilson, 1928; Roughley, 1925). Soon after
entering the oyster the worm builds two mud tubes at right angles to the edge of
the shell. The accumulated mud irritates the oyster tissue and the mollusk, in
self protection, secretes a layer of shell material over the mud tubes. A descrip-
tion of the formation of mud blisters has already been given by Whitelegge (1890)
and Lunz (1941) and need not be repeated here.
69
70 LOOSANOFF AND ENGLE
It has been the opinion of many investigators that the oysters infested with
Polydora are usually very poor. If the infestation persists, they gradually begin
to weaken and eventually succumb (Roughley, 1922; 1925). In some instances,
as for example in Australia, it has been considered advisable to grow these
mollusks on stones, logs or on specially constructed platforms, away from the
bottom. Roughley (1922, 1925) believes that the method of keeping the oysters
above the bottom mud is an effective means of preventing the infestation. It
appears that Roughley 's observations and data fully justify his conclusions in
regard to the conditions existing in Australian waters. However, recent work of
the authors carried on in Milford Harbor on the Connecticut side of Long Island
Sound, showed that some of the habits of our species of Polydora and its effects
on American oysters are somewhat different from those described for the Austra-
lian species, or previously ascribed to the mud worms common in American waters.
Description of P. Websteri Hartman
The mud worm found in the oysters of Milford Harbor was identified by
Dr. Olga Hartman of Allan Hancock Foundation, The University of Southern
California, as Polydora websteri Hartman, new name. In personal correspondence
with the authors Dr. Hartman states that the original description of the worm,
as P. caeca, was published by Webster, 1879. Since the description is faulty and
misleading in all essential respects, it has little value for systematists. Dr.
Hartman expresses an opinion that, unless caution is taken, the next reviser or
systematist is almost certain to refer to our species as the European P. ciliata,
since its morphological characters are closely akin to those of the latter. To
avoid constant confusion of Polydora websteri, which at present is a systematically
unknown species, with P. ciliata and some other species of Polydora that are
known to be very numerous in eastern America, Dr. Hartman suggested that a
description and the illustrations clearly indicating the characters of the worm
should be given in this article. In accordance with the suggestion a description
of P. websteri and the illustrations showing some of its morphological characters
are offered here. Both the description and illustrations were prepared by Dr.
Hartman.
" Polydora websteri Hartman
Polydora caeca Webster, 1879, Trans. Albany Inst., vol. 9, pp. 252-253, Figures
119-122 (not Oersted, 1843).
Polydora websteri Hartman (1942 MS on Beaufort Annelids).
The total length consists of about 105 segments and measures (preserved) 20
mm. long or shorter, but the body is usually much contracted and coiled up.
The prostomium is clearly bifid at its anterior margin; it may lack eyes or there
may be 3 or 4 weakly developed ones in trapezoidal arrangement; the prostomial
parts, palpi omitted, are shown in dorsal (Figure 1, a) and ventral (Fig. 1, b)
views. The first segment has a notopodial lobe but no notosetae, and the neuro-
podium is provided with a fascicle of slender setae. The second to fourth seg-
ments are biramous and have larger fascicles of notosetae and neurosetae with
posterior lamellae. The fifth or modified segment is longer than the others and
has, on either side, a dorsal fascicle of heavy yellow hooks with companion
POLYDORA IN OYSTERS SUSPENDED IN WATER
71
FIGURE 1. Showing certain morphological characters of P. websteri. Explanation in the text.
(Courtesy of Dr. Olga Hartman.)
72 LOOSANOFF AND ENGLE
pennoned setae, and a ventral fascicle of 5 or 6 pointed setae. The seventh
setiger has pointed setae in both fascicles. Hooded hooks are present from the
neuropodium of the eighth setiger and continued posteriorly to the end. There
are no specialized hooks in the last segments. The posterior end terminates in
a flattened collarlike disk with a dorsal notch (Fig. 1, c in posterior view) con-
siderably wider than the last few segments (Fig. 1, d in lateral view).
Branchiae, first present from the seventh setiger, are at first small but gradually
enlarge to their full size in about 5 segments; they are continued through most of
the body length but gradually decrease in size in the posterior fourth and are
absent from the last 15 or 16 segments.
The heavy hooks of the fifth setiger number about 6 projecting ones in a fas-
cicle; they are unique in that the falcate distal end has a hard, chitinous sheath
around one side; various views are shown for projecting (Figs. 1, f, g) and em-
bedded (Fig. 1, e) ones. The companion pennoned setae (Fig. 1, g) when perfect
terminate in an acute point but some may be broken off and appear frayed at
the distal ends. The hooded hooks number about 6 in a series in the middle of
the body; they have 2 well developed teeth, the major one at a right angle to the
shaft (Fig. 1, h). Tubes are fragile, constructed of silt and debris incorporated
with mucus, and occur in calcareous shells.
The original description as P. caeca Webster is incomplete in some important
details and erroneous in some others. The first segment has neurosetae, not
notosetae; the pygidium is interrupted above, not below; the companion setae of
the modified segment are pennoned, not capillary; the modified hooks of this seg-
ment are not merely falcate but have a sheath that extends some distance around
it. There may be weakly developed eyespots.
P. websteri resembles P. ciliata (Johnston) (Fauvel, 1927, Faune de France,
Vol. 16, p. 49) in some respects but the two differ in that the first has a prostomial
caruncle that extends posteriorly to the end of the third setiger and the modified
spines of the fifth setiger have a sheath around one side; in the second the pros-
tomial caruncle extends posteriorly to the second setiger and the modified spines
have an acute tooth in the concave part of the spine.
The single individual on which Webster's description was based is not known
to exist. The collection on which the present description is based is deposited
in the Allan Hancock Foundation of the University of Southern California. It
was collected from vesicles on empty oyster shells, in the mouth of the Milford
River, by Mr. J. B. Engle of the Milford Wildlife Laboratory. Since 1937 I have
obtained this species in considerable number from Beaufort, North Carolina,
Lemon Bay in southwestern Florida, and Virginia north to Connecticut. It may
be widely distributed in intertidal zones of temperate North America.
(On the plate, the small scale near the label indicates^! mm. for prostomium
and pygidium and 0.1 mm. for setal structures.)"
The authors wish to express their appreciation to Dr. Olga Hartman for the
identification of our species of the mud worm and for preparation of the description
and the illustrations of the morphological characters of P. websteri.
OBSERVATIONS
These studies were begun in April, 1940, when five large groups of oysters,
ranging from one to 5 years of age, were placed under observation in Milford
POLYDORA IN OYSTERS SUSPENDED IN WATER
73
Harbor. In the summer of the same year another group, composed of individuals
of the 1940 set, and thus being only a few weeks old, was added. Altogether over
1000 animals were used in the experiment. All these oysters were brought from
the deep water beds of Long Island Sound, where Polydora is very uncommon.
Examination of the oysters showed that only about 2 per cent of them had
mud vesicles.
Oysters of each year-class were placed on separate, large, wire trays, suspended
in the water from a float, which rose and fell with the tide. Even at low tide the
trays were at least four feet above the bottom. The oysters remained suspended
in the water until November 1, 1942. Thus, the experiment lasted 1\ years, and
FIGURE 2. Shells of an oyster infested with P. websteri. A. Cup valve. B. Flat valve.
covered two winter and three summer periods. At the end of the experiment a
random sample consisting of 20 oysters was taken for examination from each
year-class group. All the oysters were opened and the condition of their shells
and meats noted.
Examination of the shells showed that the oysters of all year-classes were
heavily infested with Polydora websteri (Fig. 2). This was true even for those of
the 1940 class which were but several weeks old when placed on the trays. The
infestation was so heavy that in main' instances separate mud vesicles could not
be distinguished. Usually the combination of several vesicles formed large mud
blisters. All the shells, with exception of one flat valve belonging to an oyster
of the 1940 class, were infested. The class of 1935, comprised of the oldest oysters,
had the greatest number of vesicles and blisters, while the youngest class had the
74
LOOSANOFF AND ENGLE
least (Table I). However, since the shells of the older oysters offered much larger
areas for infestation than those of the younger class, no direct relationship be-
tween the age of the animals and the degree of infestation could be assumed.
Such a conception was further sustained by the lack of correlation between the
age of the oysters and the degree of infestation in the other four year-classes
(Table I). In general, the cup valves of the oysters contained more vesicles and
blisters than the flat valves. This again cannot be regarded as significant because
the surface of a cup valve is considerably larger in area than that of a flat one.
Careful examination of the character and positions of the mud vesicles, and
the location of the characteristic double holes on the exterior of the shells through
TABLE I
Number of mud vesicles and blisters found in shells of oysters of different ages grown on the suspended
trays and on the bottom. Each sample consisted of 20 oysters.
TRAY OYSTERS
BOTTOM OYSTERS
YEAR CLASS
Cup valve
Flat valve
Cup valve
Flat valve
vesicles
blisters
vesicles
blisters
vesicles
blisters
vesicles
blisters
1935
208
31
177
27
13
1
17
3
1936
136
3
103
2
1937
188
20
123
17
1938
208
39
111
24
1939
189
33
156
28
1940
126
20
81
8
3
0
4
0
which the worms communicate with the outside, as well as studies of the cross-
sections of the shells clearly indicated that the infestation was not confined ex-
clusively to any one year within the experimental period. It was found, as the
result of such examination, that the infestation with Polydora began during the
summer of 1940 and continued until the end of the experiment.
While examining the shells of the oysters it was noted that in many instances
of severe infestation as many as six or seven layers of blisters, superimposing one
over the other, could be found over the same shell area. The worms occupying
the lowest, and therefore the oldest, blisters were of a larger size than those of
the upper ones. The occupants of the upper blisters were, as a rule, very small,
indicating that they entered the shell only a short time before examination. Even
under such apparently overcrowded conditions the majority of the worms were
alive and, judging by the quantities of accumulated mud, very active.
Discovering an unusually heavy infestation of the tray oysters, it was decided
to compare the degree of infestation of these animals with that of the mollusks
living on the muddy bottom. For this, samples of 20 oysters of the 1935 and
1940 year-classes were taken from the bottom of AJilford Harbor, in the area
where the float with the suspended oysters was stationed during the experiment.
Examination of the shells of the bottom oysters revealed that they were much
less infested than those kept suspended in the trays. Many bottom oysters of
the two year-classes were entirely free of mud worms. In the 1935 class, nine
POLYDORA IN OYSTERS SUSPENDED IN WATER
75
cup valves and seven flat ones bore no signs of infestation. The class of 1940 was
in even better condition, because 17 cup and 16 flat valves were entirely free of
vesicles or blisters (Table I).
In examining the condition of the oysters removed from the trays it was noted
that, regardless of the very large number of mud worms infesting their shells, the
oyster meats were in an excellent condition. They were unusually "fat," and
large in size. They appeared much superior to those of the oysters usually grown
in Milford Harbor. To verify this, a comparison was made of the experimental
oysters and the animals taken from the bottom of Milford Harbor. It consisted
in comparing the weight of the oyster meats in relation to their total weight.
Each sample consisted of 20 oysters. The results obtained indicated that the
animals suspended on the trays were much better than those collected from the
bottom (Table II). This was especially true of the oysters of the 1935 year-class,
TABLE II
Average total weight and weight of meat, and per cent of meat of oysters of different ages
grown on the suspended trays and on the bottom.
YEAR CLASS
TRAY OYSTERS
BOTTOM OYSTERS
Total
weight
Weight of
meat
Per cent of
meat
Total
weight
Weight of
meat
Per cent of
meat
1935
280.4
28.3
10.1
232.5
13.2
5.7
1936
216.2
22.1
10.2
1937
202.0
21.9
10.8
1938
154.2
17.2
11.1
1939
122.1
15.0
12.3
1940
73.1
10.1
13.7
21.8
2.4
11.0
where the bottom animals were found to be rather poor. The condition of the
bottom oysters of this age-group was further substantiated by the observations
made in connection with another series of experiments, dealing with seasonal
changes in oysters in Milford Harbor. Samples of these oysters examined on
November 15 and December 15, 1942, showed that on those dates the weight of
their meats constituted 6.5 and 5.9 per cent of their total weight.
On the basis of the above described observations the conclusion may be formed
that a heavy infestation with P. websteri does not necessarily render the oysters
poor. As was mentioned previously, the meats of heavily infested tray oysters
were in an unusually good condition. Such a condition, of course, cannot be
ascribed to commensalism with P. websteri. It indicates, nevertheless, that a
heavy infestation of their shells does not prevent oysters from becoming "fat,"
provided other environmental conditions are favorable for the existence of the
mollusks.
Regardless of the fact that the experimental oysters were suspended on the
trays, away from the bottom, they were, nevertheless, covered with a very heavy-
layer of the deposit consisting of silt, mud and various dead and alive plankton
forms. The thickness of this layer usually varied between 1/8 and 1/4 of an inch.
Such accumulation of muddy substance was more than sufficient to supply the
worms with all the mud needed for their activities. Therefore, no question could
76 LOOSANOFF AND ENGLE
be raised whether or not there was enough mud to be carried by the worms for
deposition between the shells of the oysters.
Indirectly, the experiments also provided an answer to the question of whether
or not a severe infestation with P. websteri always causes a heavy mortality among
the oysters affected. This answer is negative. For example, the most heavily
infested year-class was that of 1935. In November 1941, this group consisted
of 94 oysters. At the end of the experiment, in November 1942, 90 of these
animals were still alive. Therefore, during the last year of the experiment, when
infestation with the mud worms was presumably the heaviest, only four animals
of the total number of 94 died. Thus, the mortality for the entire year amounted
to only 4.3 per cent. This figure is considerably below that of the mortality of
oysters of the same age but living under natural conditions, where a death-rate
from 8 to 10 per cent is considered as normal.
It was also observed that a heavy infestation with mud worms did not inter-
fere with the rapid growth of the oysters. All year-classes of suspended oysters,
although heavily infested, showed a considerable increase in growth. The rate
of growth greatly exceeded that of the less infested oysters living under natural
conditions. The most noticeable difference was recorded in the case of the 1940
year-class, where at the end of two years, the average length of the suspended
oysters was 79.2 mm. as compared with 63 mm. for the bottom oysters. Inci-
dentally, our observations that the oysters kept off the bottom showed better
growth are contradictory to those of Nelson (1921) who, on the basis of his
experiments in which he also used wire trays, stated that "There was no appreci-
able difference in the rate of growth of oysters on the bottom from that of oysters
on the platform above."
DISCUSSION AND SUMMARY
It has been generally assumed that several species of Polychaete worms, such
as P. ciliata and P. ligni, are very dangerous enemies of oysters interfering with
their fattening and growth, and often causing a heavy mortality among them.
It has also been stated that a heavy infestation with Polydora can be avoided if
the oysters are grown away from the bottom mud. The method of growing
oysters off the bottom is widely used in Australia.
Results of the experiments conducted for a period of 1\ years in Milford Har-
bor, Connecticut, indicate that in this body of water certain aspects of the be-
havior of at least one species of Polydora and its effects upon infested oysters are
different from those observed in Australian waters, or ascribed to the mud worms
of certain sections of our Atlantic Coast.
The Milford experiments have shown that mud worms, Polydora websteri,
were found in much larger numbers in the shells of the oysters suspended in the
water for a period of 1\ years than in those living on the muddy bottom. This
indicates that in some areas along the Atlantic Coast of North America the suspen-
sion of oysters away from the bottom does not prevent, or eliminate, their infes-
tation with the mud worms, P. websteri. Results of the experiments also point
to the conclusion that the method of suspension may be regarded as providing
sometime more favorable conditions for the mud worms to infest the oysters.
A complete explanation as to why the mud worms preferred the tray oysters
to those on the bottom is still lacking. It may be suggested at this time, never-
POLYDORA IN OYSTERS SUSPENDED IN WATER 77
theless, that the difference in salinity at the bottom, and in the zone where the
oysters were suspended might have played an important part in the degree of
infestation of the two groups. In Milford Harbor, which is a body of water
affected by the river discharge and by inflow of salt water from the Sound, the
salinity of the upper layers of the water is usually lower than that observed near
the bottom. At times such differences are of considerable magnitude. For
example, during the rainy period of 1942 occurring in August, the salinity of the
surface layer varied between one and five parts per thousand, whereas at the
bottom the salinity remained quite steadily above 25 parts per thousand. The
fact that the heavily infested tray oysters were living in less salty water than
those existing on the bottom may indicate that P. websteri prefers the water of
considerably reduced salinity. Lunz (1941), on the basis of his observations in
South Carolina, is also of the opinion that P. ciliata is more prevalent in water of
low salinity.
The suggestion that P. websteri does not readily infest the oysters living in
water of comparatively high salinity is substantiated by the authors' examination
of oysters collected from Long Island Sound proper. During the summer of 1942
several thousand oysters of all ages were opened and examined. They were
collected from many sections of the oyster-producing area of Connecticut. Very
few oysters were found infested with Polydora. The salinity of the water of the
area from which the samples were collected is usually above 26 parts per thousand
(Loosanoff and Engle, 1940).
If certain species of Polydora, such as P. websteri, prefer water of low salinity,
it is quite possible that several outbreaks of infestation of oysters with mud worms
may be the result of prolonged rainy periods. In such cases large quantities of
fresh water entering inshore shallow areas may considerably reduce the salinity
of the water in which oyster beds are located, thus providing favorable conditions
for the spreading of Polydora infestation. Experiments on the effects of various
salinities upon the activities of Polydora, which are now being conducted by the
authors, may throw additional light upon this very interesting and important
subject.
Regardless of the heavy infestation with mud worms the meats of the tray
oysters were in a far better condition than those of the mollusks living on the
bottom. Their growth was also more rapid than that of the less infested animals
of the same ages, but living under natural conditions. These two observations
indicate that a heavy infestation with P. websteri does not necessarily interfere
with the feeding and fattening of oysters, nor impair their growth. The apparent
lack of ill effects upon the growth and fattening of oysters can be easily under-
stood, if it is remembered that Polydora is not a parasite. Each worm remains in
contact with the fleshy tissues of the oysters for a comparatively brief period.
As soon as the mollusk covers the intruder and its mud tubes with a layer of shell
material, the worm becomes isolated and cannot exert toxic effects upon the
tissues of the oyster. It is probable, nevertheless, as Lunz (1941) indicated, that
a large number of mud blisters within the shell may restrict the living space of
the oyster, and that the animal may be forced to spend considerable energy in
secreting the shell material for covering the mud worms. It is also possible that
large quantities of mud accumulated by the worms on the bottom may render
LOOSANOFF AND ENGLE
the environmental conditions unfavorable for- the existence of the oysters and
may even cause a heavy mortality among those mollusks (Roughley, 1922).
Milford experiments have also shown that a severe infestation with P. websteri
did not cause a heavy mortality of the oysters. Our observations coincided with
those of Lunz (1941) on P. ciliata who found that "In the five year period during
which these pests have been under observation in South Carolina and other south-
ern states, no high mortality has been found on oyster beds which could be
attributed to the activities of Polydora."
LITERATURE CITED
FIELD, IRVING A., 1922. Biology and economic value of the sea mussel, Mytilus edulis. Bull.
U. S. Bur. Fish., 38: 128-259.
KAVANAGH, L. D., 1940. Mud blisters in Japanese oysters imported to Louisiana. Louisiana
Conservation Review for Autumn, 1940: 31-34.
LEBOUR, M. V., 1907. The mussel-beds of Northumberland. Northumberland Sea Fisheries
Committee. Report on the Scientific Investigations for the year 1906: 28-46. New Castle-
on-Tyne.
LOOSANOFF, VICTOR L., AND JAMES B. ENGLE, 1940. Spawning and setting of oysters in Long
Island Sound in 1937, and discussion of the method for predicting the intensity and time
of oyster setting. Bull. U. S. Bur. Fish., 74: 217-255.
LUNZ, G. R., JR., 1940. The Annelid worm, Polydora, as an oyster pest. Science, 92: 310.
LUNZ, G. R., JR., 1941. Polydora, a pest in South Carolina oysters. Journ. of the Elisha Mitchell
Scientific Society, 57: 273-283.
NELSON, T. C., 1921. Report of the department of biology of New Jersey Agricultural College
Experiment Station for the year ending June 30, 1920. New Jersey Agricultural Ex-
periment Station, 1919-1920: 317-349.
NELSON, THURLOW C., AND LESLIE A. STAUBER, 1940. Observations of some common Polychaetes
on New Jersey oyster beds with special reference to Polydora. Anat. Rec., 78: 102.
ROUGHLEY, T. C., 1922. Oyster culture on the George's River, New South Wales. Technical
Education Series, No. 25, Technological Museum, Sydney, 1-69.
ROUGHLEY, T. C., 1925. The story of the oyster. Australian Museum Magazine, 2: 1-32.
TAKAHASHI, KEIZO, 1937. Notes on the polychaetous annelid Polydora pacifica n. sp. which bores
holes in Pinctada margaritifera (Linne). Palao Trop. Biol. Stat. Studies, 1: 155-167.
WHITELEGGE, T., 1890. Report on the worm disease affecting the oysters on the coast of New
South Wales. Records of the Australian Museum, 1: 41.
WILSON, DOUGLAS, P., 1928. The larvae of Polydora ciliata Johnston and Polydora hoplura
Claparede. Jour. Mar. Biol. Ass'n N. S., 15: 567-603.
THE ACTION OF ACETYLCHOLINE ON THE ISOLATED
HEART OF VENUS MERCENARIA
ROBERT B. WAIT
(Biological Laboratories, Harvard University, Cambridge)
INTRODUCTION
The extraordinary sensitivity of the heart of the lamellibranch mollusc,
Venus mercenaria, to acetylcholine was first reported by Prosser and Prosser
(1937). Smith and Levin (1938) suggested the use of the isolated heart as a test
object for acetylcholine and indicated its very much greater sensitivity to acetyl-
choline than to choline. The first detailed account of the responses of the Venus
heart to acetylcholine and to nerve stimulation was by Prosser (1940). Prosser
presented evidence that nervous inhibition of the heart is probably due to the
release of acetylcholine at the terminations of nerve fibers from the visceral
ganglion.
With the idea of using the isolated Venus heart for determining the acetyl-
choline content of tissues, when only small amounts are available, further experi-
ments were carried out to ascertain the nature of the concentration-action curve
and the imporatance of temperature control. The results will be reported briefly.
METHODS
Supplies of animals were obtained from a local market and stored dry at 5° C.
until used. They ordinarily remained in a satisfactory condition for one to two
weeks.
Certain minor changes in the method suggested by Prosser (1940) for isolating
and perfusing the heart were made, hence the general procedure will be outlined.
The soft parts were exposed dorsally by breaking and removing the umbos and
hinge of the valves. The mantle and pericardium overlying the heart were cut
away exposing the single, median ventricle and the laterally-disposed, thin-
walled auricles. A thread was passed under each auricle and tied at the junction
with the ventricle. The auricles were cut distal to the threads, also the anterior
and posterior blood vessels and the intestine which passes through the heart.
The isolated ventricle (which will be spoken of as the "heart") was placed in a
bath with a capacity of 10 or 20 cc. when filled to the overflow arm. This was
supplied with a common inlet-outlet tube at the bottom for perfusion fluid and an
additional inlet for air, needed mainly for stirring since the oxygen requirements
of the heart are low. This arrangement is shown in Figure 1. When temperature
regulation was desired this chamber was submerged to the overflow arm in a
water bath, the temperature of which could be kept constant or varied as desired.
The heart was attached to a light heart lever counterweighted to 250 mg. and the
beat recorded on a slow kymograph. The advantage of suspending the heart by
the auricles is the avoidance of interference by the short length of intestine which
79
80
ROBERT B. WAIT
passes through the longitudinal axis of the ventricle, the amplitude of beat being
greater and more constant than when the heart is suspended by the anterior and
posterior ends.
| ^ Since the blood of Venus mercenaria is very similar in composition to sea water
(Cole, 1940) the latter was used as a perfusion fluid with quite satisfactory results.
Glucose was added to the sea water in the proportion of 0.25 grams/liter. Isolated
hearts have been kept beating for as long as three days at 15° C. During periods
PERFUSATE
OUTLET
B
FIGURE 1. Perfusion chamber showing arrangement of air inlet and common inlet-outlet for
perfusion fluid. Spring clamps at A and B, when opened and closed alternately, allow washing
without complete removal of fluid from the chamber.
of washing the level of fluid in the bath was not allowed to drop below the level
of the heart as such mechanical disturbance often causes temporary cessation of
beat.
The acetylcholine used was in the form of the chloride and a stock solution
was made up of 10~3 by weight of the alkaloid in 5 per cent NaH2PO4. This has
a pH of approximately 4.0, at which acetylcholine is quite stable. The solution
was sealed in small ampoules which were heated at 100° C. in a water bath for
five minutes and then stored in a refrigerator until needed. Just before using,
the stock acetylcholine was diluted with sea water so that a series of dilutions
were at hand from which known amounts, up to one cc., when added to the bath,
gave the desired concentration. The acetylcholine was added at the bottom of
the bath by means of a hypodermic syringe with a long, small bore, glass tube
bent at a right angle, in place of a needle. When the acetylcholine was added a
corresponding volume of sea water was automatically displaced from the top of
the bath before any appreciable mixing by the stream of air bubbles occurred.
ACETYLCHOLINE ACTION ON VENUS HEART 81
In a given test the acetylcholine was left on the heart for one minute during
which time the amplitude reached a new and nearly constant level. The heart
was then washed with several changes of sea water and allowed a period of five
minutes to recover its original amplitude before a second test was made. - Meas-
urement of the amplitude before and near the end of a given test allowed calcula-
tion of the amount of inhibition resulting from the action of the drug on the heart,
and this was taken as a measure of effect.
Although eserine increases the sensitivity of the Venus heart to acetylcholine
it was not used due to the fact that recovery between tests is more rapid in the
non-eserinized heart.
RESULTS
1. The concentration-action curve for acetylcholine inhibition.
Besides any theoretical significance in the quantitative relation between the
concentration of a drug and its effect on a given biological system it is of practical
importance in bioassay to know the nature of the concentration-action curve for
the particular drug and the preparation being used. If it is not a straight line
one can select that range where the response shows the greatest change with
small differences in the concentration of the substance under investigation.
Clark (1933) has pointed out that for most potent drugs such as acetylcholine,
adrenaline, histamine and nicotine the concentration-action curves follow a
hyperbola and that, depending on the kind of preparation and the recording sys-
tem, responses are sometimes less accurately measured near the threshold and in
other cases as they approach 100 per cent. It will be seen in the case of the Venus
heart that the responses are most accurately determined in the vicinity of 50
per cent inhibition.
Concentration-action curves were obtained for 15 isolated Venus hearts. A
series of sample records of the responses of one of these hearts is shown in Figure 2.
From such records the per cent inhibition for each concentration could be meas-
ured and, in each case, when the results were plotted the curve was a hyperbola.
Due allowance had to be made in some instances for the apparent complete in-
hibition of the heart before the flat portion of the curve was attained. This was
due to the inertia in the recording system and the consequent failure of the record
to show small residual movement.
In most preparations commonly used in the assay of acetylcholine, such as the
isolated frog heart, the frog rectus abdominis and the dorsal muscle of the leech,
the range over which graded action is obtained is from 1000 to 10,000 fold (Clark,
1933). In the case of the isolated lobster heart graded effects may be obtained
over a 1,000,000 fold range (Welsh, 1942). In such cases it is customary to plot
a measure of the effect against the logarithm of the concentration. Such curves
are always S-shaped. When the amount of inhibition of the Venus hearts was
plotted against the logarithm of the concentration most of the curves were such
as is seen in Figure 3, which is that of a typical heart. This curve which was
drawn to fit the points emphasizes the difficulty of making accurate measurements
as the responses approach maximum. Since it is also difficult to determine
accurately small amounts of inhibition it is obvious that in using the Venus heart
for bioassay it is better to choose such concentrations of knowns and unknowns
that the amount of inhibition produced is between 20 per cent and 80 per cent.
82
ROBERT B. WAIT
ACH 2X10'
ACH 5X 10"
ACH 9XIO
"'2
ACH 10
ACH 2X10"
ACH 7X10
-ii
ACH 9X10"
ACH I 0
"'°
ACH 2X 10
.-10
ACH 2.5 X 10
FIGURE 2. Sample kymograph records from a series on one heart showing graded action of
acetylcholine over a range from threshold to complete inhibition.
ACETYLCHOLINE ACTION ON VENUS HEART
83
2. Effect of temperature on the response to acetylcholine.
Some of the concentration-action curves were obtained in February, others
as late as May. During February the concentrations of acetylcholine which
produced a just measurable inhibition on different hearts were between 5 X 10~12
(1 : 5,000,000,000,000) and 5 X lO"11. During May thresholds were found as
high as 5 X 10~10. This was at first thought to be evidence of a seasonal varia-
tion in sensitivity, although Prosser (1940) reported the highest sensitivity to
100 -
CD
X
z
LJ
o
ac
ui
a.
20 -
5X10
10
5X10
CONC. ACH.
10
5X10
FIGURE 3. Data from a typical heart showing the decrease in amplitude (per cent inhibition)
as a function of the concentration of acetylcholine (ACh).
occur in the spring. The experiments done thus far has been at room tempera-
ture and there was some evidence that the sensitivity of a heart was lower when
the room temperature was abnormally high. Therefore a few experiments were
performed to determine the effect of temperature on the response of the heart to
acetylcholine. By means of a bath, with temperature control, the chamber
containing the heart, and the perfusion fluid, could be maintained at any tem-
perature between 5° and 35° C. The range over which hearts were observed to
beat satisfactorily was somewhat less than this. Beginning in some cases at a
low temperature and in others at a high, the concentration of acetylcholine was
found which would produce a 50 per cent decrease in amplitude. The tempera-
ture was then increased or decreased and after a period of adaptation the con-
centration of acetylcholine necessary for 50 per cent inhibition was again de-
84
ROBERT B. WAIT
termined. The results on three hearts, which had approximately the same thresh-
old sensitivity at a given temperature, are shown in Figure 4. That there is a
marked effect of temperature on the response of a given heart to acetylcholine is
apparent. Approximately 100 times as much acetylcholine is required to produce
50 per cent inhibition at 30° C. as is required at 10° C. For this reason, and also
from a consideration of the average environmental temperature of Venus mer-
cenaria, it may be concluded that 15° C. is a satisfactory temperature at which to
30
a.
2
LU
20
10
Id"
10'°
l<59
CONC. ACH.
id*
I07
FIGURE 4. Data from three hearts, each represented by a different symbol, showing the
concentration of acetylcholine (ACh) necessary to produce 50 per cent inhibition at different
temperatures.
maintain the isolated venus heart for use in bioassay. If temperature control
is not employed it is obviously necessary to perform a given set of assays at a
nearly constant temperature.
It is probable that the increase in the amount of acetylcholine required to
produce a given amount of inhibition, as the temperature is raised, is due to the
activation of the enzyme cholinesterase which destroys acetylcholine, and which
is present in the Venus heart in small amounts (Jullien, et al, 1938; Smith and
Click, 1939).
CONCLUSIONS
This further study of the response of the isolated heart (ventricle preparation)
of Venus mercenaria to acetylcholine provides information which confirms and
extends that of Prosser (1940). Since the work was done with the practical view-
point of eventual use of the preparation in assaying for acetylcholine in tissue
extracts, little attention has been directed toward certain interesting theoretical
ACETYLCHOLINE ACTION ON VENUS HEART
problems. The demonstration that the concentration-action curve is a hyper-
bola, and recognition of the difficulty of recording beats of small amplitude,
indicates that determination of acetylcholine. values can most accurately be made
when the concentrations are such as to produce between 20 and 80 per cent de-
crease in amplitude.
The importance of temperature control is evident. A heart which is rela-
tively insensitive to acetylcholine at 25° to 30° C. becomes 100 times more re-
sponsive at 5° to 10° C. A temperature midway in this range has been found to
preserve a beat of satisfactory amplitude and frequency for a convenient length
of time (12 to 24 hours).
LITERATURE CITED
CLARK, A. J., 1933. The mode of action of drugs on cells. Edward Arnold and Co., London.
COLE, W. H., 1940. The composition of fluids and sera of some marine animals and of the sea
water in which they live. Jour. Gen. Physiol., 23: 575-584.
JULLIEN, A., D. VINCENT, M. BOUCHET AND M. VIULLET, 1938. Observations sur 1'acetylcholine
et la choline-esterase du coeur des Mollusques. Annales de Phvs. et de Phvs. Biol., 14:
567-574.
PROSSER, C. L., 1940. Acetylcholine and nervous inhibition in the heart of Venus mercenaria.
Biol. Bull., 78: 92-102.
PROSSER, C. L., AND H. B. PROSSER, 1937. The action of acetylcholine and of inhibitory nerves
upon the heart of Venus (abstract). Anal. Rec., 70, Sup. 1: 112.
SMITH, C. C., AND D. CLICK, 1939. Some observations on cholinesterase in invertebrates (ab-
stract). Biol. Bull, 77: 321-322.
SMITH, C. C., AND L. LEVIN, 1938. The use of the clam heart as a test object for acetylcholine
(abstract). Biol. Bull., 75: 365.
WELSH, J. H., 1942. Chemical mediation in Crustaceans. IV. The action of acetylcholine on
isolated hearts of Homarus and Carcinides. Jour. Cell, and Conip. Physiol., 19: 271-279.
Vol. 85, No. 2 October, 1943
THE '
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE HISTOGENESIS AND CYCLIC PHENOMENA OF THE
SINUS GLAND AND X-ORGAN IN CRUSTACEA
ROBERT W. PYLE
(Woods Hole Oceanographic Institution 1 and Department of Biology, Rensselaer
Polytechnic Institute, Troy, New York)
\
INTRODUCTION
In the decade that has followed Hanstrom's (1933, 1934) description of the
sinus gland and X-organ in Crustacea a number of investigators, Sjogren (1934),
Hanstrom (1937), and Stahl (1938) have described them in detail. All of these
studies have been concerned with a description of these glands as they appear
in the adult animal. Since there has been little or no work done upon the
histogenesis of either the sinus gland or X-organ, it is one of the objects of this
paper to describe the histogenesis of both the sinus gland and X-organ in detail.
The endocrine activity of the sinus gland has been more or less well established
through numerous studies in the past several years. As these are quite ade-
quately and critically examined by Scharrer (1941) and Kleinholz (1942) there is
no need to review the literature in detail. For further information of this nature
one should examine those papers. Although extensive physiological studies have
been made in relation to the endocrine function of the sinus gland, there have
been no cytological studies made (except in Cambarus by Dethier 1942) to deter-
mine whether or not there are any evidences of cyclic phenomena in this gland.
The role of the X-organ has been suggested, but no cytological studies have been
made of it. Both the X-organ and sinus gland have been cytologically examined
and the results of this study are reported herein.
METHODS AND MATERIALS
The histogenesis of the sinus gland and X-organ were studied in two species
of Crustacea, Homarus americanus and Pinnotheres maculatus. The adults of
these species and of Cambarus virilis were studied for cytological evidences of
cyclic phenomena during the moulting period.
The eggs of Homarus were fixed in Carnoy-Lebrun : the first four stages after
hatching were fixed in Zenker-formol and Bouin-Duboscq-Brasil, and the adult
eye stalks (one week, 48 hours, six hours before, during, six hours, 48 hours, one,
1 Contribution No. 326 from Woods Hole Oceanographic Institution.
87
ROBERT W. PYLE
one and one-half, four, six and thirteen months after moulting) 2 were fixed in
Zenker, Zenker-formol and Bouin-Duboscq-Brasil. The eggs and first four
stages after hatching were doubly imbedded in parlodion and paraffin and sec-
tioned at five to nine micra. In some of the adults the exoskeleton of the eye
stalk was decalcified and the whole eye stalk was doubly imbedded in parlodion
and paraffin and sectioned at seven to 12 micra. In other adults the exoskeleton
of the eye stalk was removed and the specimens were singly imbedded in paraffin.
These were sectioned at seven to 15 micra.
The eggs, first zoea and adult stages (before, during, after and between
moulting periods) of Pinnotheres were studied. The eggs and first zoea were
fixed in Carnoy-Lebrun, Zenker-formol and Bouin-Duboscq-Brasil, and were
doubly imbedded in parlodion and paraffin. Sections were cut at four to seven
micra. The various stages of the adult were fixed in Zenker-formol and Bouin-
Duboscq-Brasil, and were doubly and singly imbedded and sectioned at six to
12 micra.
The eye stalks of Cambarus were treated in the same way as those of Homarus;
some were singly and some doubly imbedded. Sections were cut at seven and
nine micra.
Serial sections were made of all specimens and these were stained with
haemalum and eosin, Mallory's triple, Foot's (1933) and Lillie's modifications of
the Masson trichrome stain, and the protargol method of Bodian (1937).
OBSERVATIONS
A. Pinnotheres maculatus
The X-organ is found in the embryo just before hatching (Figs. 1, 19) in that
part of the eye which will become the median ventral side of the eye stalk in the
PLATE I 3
The histogenesis of the sinus gland and the X-organ in Pinnotheres maculatus. All figures
are oblique frontal sections of the right eye stalk. The neuropile of the optic ganglion is white
and the ganglion cell layer is stippled.
FIGURE 1. Section of the late egg stage showing the position of the X-organ in relation to
the structures of the optic ganglion. X — X-organ. S. G. — Sinus gland.
FIGURE 2. Section of the first adult stage showing the positions of the sinus gland and
X-organ: both are distal to the medulla terminalis.
FIGURE 3. Section of the second adult stage. The sinus gland has begun to move distally,
but the X-organ is found in the same general position as in earlier stages.
FIGURE 4. Section of the third adult stage. The sinus gland has advanced to a point be-
tween the medulla interna and medulla externa.
FIGURE 5. Section of the fourth adult stage. The sinus gland is lateral to the medulla
externa at this stage.
FIGURE 6. Section of the fifth adult stage. The sinus gland now occupies a position between
the medulla externa and lamina ganglionaris.
FIGURE 7. Section of the sixth adult stage. The sinus gland has advanced to a point that
is distal to the lamina ganglionaris. In this as in previous stages the X-organ is found distal to
the medulla terminalis.
2 I am indebted to Dr. Charles J. Fish and the staff at the Wickford Hatcheries for the deter-
mination of the times in those specimens that were about to moult.
3 All figures have been drawn with the aid of a micro-projection apparatus. All structures
found between the hypodermis of the exoskeleton and the optic ganglia have been omitted for
the sake of clarity.
SINUS GLAND AND X-ORGAN IN CRUSTACEA
89
SG
7
PLATE I
90 ROBERT W. PYLE
first zoea. It occupies a position on the distal portion of the medulla terminalis
and is almost entirely surrounded by the cells of the optic ganglia. There is
also found, usually between the X-organ and the medulla terminalis, an area
which is devoid of cells; this appears in serial sections as a hole. As far as can
be determined from cytological preparations the cells of the X-organ are very
similar to those found in the ganglion cell layer. They are probably derived from
the same embryonic source and later become differentiated into X-organ cells.
There is no evidence of any nerve fiber connections with the medulla terminalis.
The nuclei are of the same size, shape and appearance as the nuclei of the ganglia
cells. There is more cytoplasm present than in the ganglia cells; it is non-
granular and clear. The X-organ is an integral part of the ganglia cell layer and
is not set apart from it by a connective tissue sheath.
The secretory products of the X-organ are large rounded masses which exhibit
concentric rings; this seems to indicate that the secretions have been laid down at
different intervals. These secretory products always give a basophilic reaction
when stained; they are blue after aniline blue and are structurally very similar
to those found in the X-organ of Homarus. There is no evidence of any cyclic
phenomena in the egg stage as the secretions have the same characteristic appear-
ance in all specimens.
There is no evidence that the sinus gland has been developed by the late egg
stage or the first zoeal stage. Unfortunately, conditions existing at Woods Hole
last summer did not permit obtaining the intermediate forms between the first
zoeal and the first adult stage so that these could not be studied. Attempts to
raise them beyond the first zoeal stage were fruitless.
The adults may be grouped into six categories or stages which correspond to
the moults. This is comparable to the five moults found in Pinnotheres pisum by
Atkins (1926). In all stages of the adult (Figs. 2-7, 21) the X-organ is found in
the same relative position that it occupies in the egg stage. The number of
cells of which it is composed increases after each moult, but no mitoses were
observed at any time. The cells are larger than the ganglia cells which surround
them. They are wedge-shaped and are grouped in such a way as to remind one
(when examining serial sections) of a pie that has been cut; the nuclei are found
around the periphery and each cell becomes narrower as its cytoplasm extends
toward the center of the X-organ. As the cytoplasm becomes filled with the
secretory products the nucleus is pushed more and more toward the periphery
PLATE II4
The histogenesis of the sinus gland and X-organ in Homarus americanus. All figures are
oblique frontal sections; figures 8, 9, and 11 are of the left eye stalk, and figure 10 is of the right
eye stalk. The neuropile of the optic ganglion is white and the ganglion cell layer is stippled.
FIGURE 8. Section of the late egg stage showing the position of the X-organ.
FIGURE 9. Section of the third stage after hatching. The sinus gland is seen as a thin
structure on the proximal side of the medulla externa.
FIGURE 10. Section of the fourth stage after hatching. The sinus gland still occupies a
position on the proximal side of the medulla externa. The X-organ extends to the hypodermis
of the exoskeleton where the eye papilla is found.
FIGURE 11. Section of the adult stage. Both the X-organ and sinus gland, on opposite
sides of the eye stalk, are seen extending beyond the limits of the neurilemma which surrounds the
optic ganglion.
4 See footnote 3.
SINUS GLAND AND X-ORGAN IN CRUSTACEA
91
5
OH
00
92 ROBERT W. PYLE
of the cell. In no stage is there any evidence that the X-organ has a nerve fiber
tract which extends from it to the medulla terminalis. Cytologically there is no
evidence of any cyclic phenomena associated with the secretion processes of the
X-organ at any time. It always exhibits the same basophilic reaction regardless
of whether it is fixed before, during, after or between moulting periods.
The sinus gland is well developed by the time the animal reaches the first
adult stage (Fig. 2). It is at this time that it enters the mussel, Mytilus edulis,
and begins its parasitic form of existence. The most remarkable feature in the
subsequent development of the sinus gland is its change of position in relation
to the structures of the optic ganglia. In the first stage (Fig. 2) it is found closely
appressed to the medulla terminalis; in the second stage (Fig. 3) it has begun to
move away from the medulla terminalis. In each successive stage it advances
farther toward the distal portion of the eye stalk. In the third stage (Fig. 4)
it occupies a position between the medulla interna and the medulla externa, in
the fourth stage (Fig. 5) alongside the medulla externa, in the fifth stage (Fig. 6,
21) between the medulla externa and lamina ganglionaris, and in the sixth stage
(Fig. 7) it has advanced to a point that is distal to the lamina ganglionaris.
In all stages the sinus gland is found on the dorso-lateral side of the eye stalk.
There are very few nuclei in it (Fig. 23) and these bear such a close resemblance
to those of the neurilemma, which is continuous with the sinus gland, that
one might well consider the sinus gland a modification of the neurilemma (cf.
PLATE III5
Microphotographs to show the cyclic phenomena in the sinus gland of Homarus americanus
and Cambarus virilis. All photographs are X 725 reduced about 35 percent, and are of materials
fixed in Bouin-Duboscq-Brasil and stained with Foot's modification of the Masson trichrome
stain.
FIGURE 12. Homarus americanus. A portion of the edge of one lobule of the sinus gland
of a specimen fixed forty eight hours before moulting and sectioned at twelve micra. The
brilliantly staining acidophilic secretory products are seen as dark irregularly shaped masses.
This and succeeding figures show the loose network of connective tissue which constitutes the
framework of the gland and the very few scattered nuclei.
FIGURE 13. Homarus americanus. A portion of the edge of one lobule of the sinus gland
of a specimen fixed six hours after moulting and sectioned at ten micra. The bulk of the secretory
masses are only slightly acidophilic and appear less dark in the photograph.
FIGURE 14. Homarus americanus. A portion of the edge of one lobule of the sinus gland
of a specimen fixed six months after moulting and sectioned at twelve micra. The secretory
material is reduced in quantity and stains in a slightly acidophilic manner.
FIGURE 15. Cambarus virilis. • A portion of the sinus gland of a specimen fixed before
moulting and sectioned at nine micra. The numerous brilliantly acidophilic secretory masses
are seen as dark masses hung upon the connective tissue framework of the gland. The blood
sinus shows as clear areas.
FIGURE 16. Cambarus virilis. A portion of the sinus gland of a specimen fixed after moulting
and sectioned at nine micra. The secretory products are conspicuous by their absence. The
blood sinuses are filled with blood. The nuclei are scattered at random in the loosely arranged
connective tissue.
FIGURE 17. Cambarus virilis. A portion of the sinus gland of a specimen fixed in December
and sectioned at nine micra. The majority of the secretory products present are acidophilic.
The blood sinuses appear as clear areas.
6 All microphotographs were made using Bausch and Lomb microphotographic equipment.
The photographs were taken on Eastman Super Panchro Press film, and were printed on Eastman
Azo F-2, and Velour Black S-4 paper. Wratten filters G No. 15 and X-l were used.
SINUS GLAND AND X-ORGAN IN CRUSTACEA
93
' fft
4V
•V 19
Y * :
&
• 0*
*j *
14 '•
•>
in
&
*
>
0
4
(^
.v
16
t 17 i*
PLATE III
94 ROBERT W. PYLE
Hanstrom, 1939). In no stage of the adult is the sinus gland more than partially
extruded beyond the level of the neurilemma. In all stages after the second, one
finds a very large bundle of nerve fibers passing from the sinus gland toward the
medulla terminalis. Some of these pass directly into the neuropile of the medulla
terminalis and some ramify among the adjacent ganglion cells. In the first two
adult stages the sinus gland is too closely appressed to the medulla terminalis
for the presence of the nerve fibers to be readily determined. There is not, how-
ever, any evidence that there is a nerve fiber tract which extends directly to the
brain as has been reported in Cambarus by Welsh (1941).
There are no obvious cell boundaries in the sinus gland. In fact there are
so few nuclei to be found in any particular specimen's sinus gland that the indi-
vidual cells which constitute the gland must be relatively very large. It is
possible to detect canals which extend toward the blood sinus of the eye stalk;
presumably these carry the secretory products to the blood stream. The secre-
tions are in the form of large, more or less irregular, masses the amount of which
varies very little regardless of the nearness or remoteness of the moulting period.
The secretions of the sinus gland give a basophilic reaction to the stains em-
ployed before, after and in the intermoult periods. In specimens fixed while in
the process of moulting that portion of the sinus gland which is next to the
neuropile of the adjacent optic ganglion gives an acidophilic reaction, whereas,
PLATE IV «
Microphotographs to show the sinus gland and X-organ in Homarus americanus and Pin-
notheres maculatus. Figures 18, 19, 20, 22, 23 X 725, and figure 21 X 150; all are reduced about
25 percent. The material shown in figure 18 was fixed in Carnoy-Lebrun, that of figure 20 was
fixed in Zenker-formol, and all others were fixed in Bouin-Duboscq-Brasil. The material shown
in figure 22 was stained with Mallory's triple stain, and all others were stained with Foot's modifi-
cation of the Masson trichrome stain.
FIGURE 18. Homarus nniericanus. \ portion of the optic ganglion of an embryo fixed in
the late egg stage and sectioned at five micra. The arrow indicates the characteristic secretory
products of the X-organ which is surrounded by the cell layer of the optic ganglion. (Compare
with Fig. 8.)
FIGURE 19. Pinnotheres maculatus. Section of the late egg stage embryo showing the
position (arrow) of the X-organ. Sections were cut at four micra. (Compare with Fig. 1.)
FIGURE 20. Homarus inner ican-us. A portion of the eye stalk of a fourth stage embryo,
sectioned at seven micra, showing the close association of the X-organ with the cells underlying
the eye papilla. The bulge in the exoskeleton can be noted at the top of the photograph. Note
that there are fewer nuclei in the X-organ, per unit area, than in the adjacent optic ganglion.
(Compare with Fig. 10.)
FIGURE 21. Pinnotheres maculatus. Section at eight micra of the eye stalk showing the
general relationship of the various structures found therein. (Compare with Fig. 6.)
FIGURE 22. Homarus americanus. Section of the eye stalk of a third stage after hatching
specimen (at seven micra) which shows the sinus gland lying just above the deeply staining
muscle. Note that it stains much as the surrounding ganglion does and that the blood sinus is
quite small.
FIGURE 23. Pinnotheres maculatus. Section of the sinus gland shown in figure 21 enlarged
to show its structure. This is the gland of a specimen that had been starved for forty-six days.
Only one nucleus is to be found in this section, and what few secretory products are seen are stained
brilliantly acidophilic. Note the indefiniteness to the connective tissue framework of the gland.
6 All photographs, excepting that of figure 20, were taken using the same equipment and
materials that were used for those of Plate III. The photograph for figure 20 was made on East-
man Ortho-X film using only the X-l Wratten filter.
SINUS GLAND AND X-ORGAN IN CRUSTACEA
95
. v
•. ;
•"*' •**» '25'LTC
20
i
• t
s
23
PLATE IV
96 ROBERT W. PYLE
the lateral portion, next to the blood sinus, gives a basophilic reaction. The
acidophilic and basophilic portions blend together in the middle of the gland.
Some specimens were starved for varying lengths of time. Those which had
been starved for eight days exhibit both an acidophilic and basophilic reaction,
but the two reactions are not regionally differentiated as is the case in specimens
fixed while in the process of moulting. This reaction is found regardless of
whether the specimens are fixed before, during or after the moulting period. In
specimens starved for as long as 46 days (Figs. 21, 23) one finds only an acidophilic
reaction regardless of the nearness or remoteness of the moulting period. Like-
wise, as the period of starvation is increased the amount of secretory material
present in the gland is decreased although there is no evidence that the decrease
due to starvation affects the frequency of moults in this particular animal.
B. Homarus americanus
The X-organ is found in the late egg stage (Figs. 8, 18); it is comparable in
appearance to the X-organ in Pinnotheres, although there are definite structural
differences in it. It is located in that part of the eye stalk that will become the
median somewhat ventral side in the first stage after hatching. It is entirely
surrounded by the cellular layer of the adjacent optic ganglion, but is separated
from the ganglia cells by a thin connective tissue sheath. A definite bundle of
nerve fibers extends from the X-organ to the medulla terminalis. The nuclei of
the X-organ cells are histologically the same as those of the surrounding ganglia
cells: the cytoplasm is more abundant than in the ganglia cells, and that which
does not contain secretory products is clear and stains lightly. The secretory
products show a series of concentric layers, when sectioned, comparable to those
found in Pinnotheres; the nuclei are pushed to one side by the secretory products
which nearly fill the entire cytoplasm. In all stages, under low power of the
microscope, the X-organ has a similar appearance. This characteristic appear-
ance has been described by Hanstrom (1939) as a "bunch of grapes." The distal
portion of the X-organ extends to the median somewhat ventral portion of the
hypodermis of the eye stalk. There is no evidence that there is any eye papilla
formed at this time; in later stages the association of the X-organ and the eye
papilla is evident. The X-organ exhibits no cyclic phenomena, cytologically,
in the egg. The secretory products are always basophilic to the stains employed
and vary very little in quantity.
In the first four stages after hatching (Figs. 10, 20) the X-organ increases
greatly in size; this is due to the greatly increased number of cells in it and the
increased amount of secretory products. Although there is a large increase
in the number of cells found in the X-organ there is evidence of only an occasional
mitosis after the animal has hatched. At its distal portion the X-organ conies
into close contact with the exoskeleton which is bulged at this point. The cuticle
of this particular region is extremely thin; this is the eye papilla (Figs. 10, 20).
The eye papilla cells are found on the distal side of the X-organ between it and
the ommaticlia of the eye. There is no connection between the X-organ and the
eye papilla as the X-organ is completely surrounded by a connective tissue sheath.
No bipolar cells are found in the distal portion of the X-organ that are com-
parable to those described by Hanstrom (1937, 1939) for the adult of Homarus
SINUS GLAND AND X-ORGAN IN CRUSTACEA 97
americanus. Cytologically there is no evidence of any cyclic phenomena in the
X-organ during the first four stages after hatching; the secretory products are
basophilic and the quantity is quite constant.
In the adult (Fig. 11) the X-organ no longer extends to the exoskeleton, but
is found in the proximal half of the eye stalk. The basal portion of it is imbedded
in the cellular layer of the distal part of the medulla terminalis. The distal
portion extends well beyond the ganglionic cellular layer (to a point approxi-
mately level with the distal end of the medulla interna) into the blood sinus of
the eye stalk. The X-organ occupies the same general position that it occupies
in the earlier stages. In general structure it has become considerably more com-
plex; it is now divided into a large number of units each of which is composed of
from ten to twenty or more cells. Each of these units has a circular, whorled
appearance. The nuclei are arranged around the periphery and the secretory
products occupy the central area. A large bundle of nerve fibers passes around
each of the units giving off nerve fibers to the individual cells. This arrangement
gives the serial sections an appearance of being a series of whorls each of which
originates from a common central stem of nerve fibers. The main bundle of
nerve fibers passes between the various units and extends to the median side of
the medulla terminalis. The nuclei have increased prodigiously in numbers,
but still bear a marked resemblance to those of the cells of the optic ganglia.
The cytoplasm of the X-organ cells is large and irregular in shape; it is filled for
the most part with secretory products which have the characteristic concentric
layers within them. Cells not possessing secretions have a clear lightly staining
cytoplasm.
There is no evidence of cyclic phenomena associated with moulting as far as
the X-organ is concerned. The basophilic reaction is found regardless of whether
the eye stalk has been fixed a few days, 48 hours, six hours before, six hours,
48 hours, one, one and one-half, four or six months after moulting. Likewise,
there is little change in the amount of secretory products that are evident in the
X-organ of the eye stalks in the above series; the number of blue staining con-
cretions is remarkably constant. In the case of the specimen that had not
moulted for more than one year there were fewer secretory products present and
more of the units contained vacuoles.
As far as can be determined the sinus gland is not formed sufficiently to be
definitely recognized as such until the third stage after hatching (Figs. 9, 22).
At this time it is a thin, lightly staining structure located on the dorso-lateral side
of the eye stalk between the medulla interna and medulla externa. It is not very
conspicuous as it does not give the typically brilliant acidophilic reaction to acid
fuchsin that is found in the adult sinus gland. Structurally the sinus gland has
the appearance of being a thickened portion of the neurilemma which invests
the optic ganglia. The nuclei are few in number and stain precisely in the same
manner as the nuclei of the neurilemma. The cell boundaries cannot be dis-
cerned ; the cytoplasm seems to be confined to the connective tissue framework
of the gland upon which the secretory materials are hung. The general tissue
of the gland, regardless of what it is composed, stains very lightly with all the
stains employed. There is a definite nerve fiber tract which extends from the
sinus gland to the lateral distal border of the medulla terminalis. It is this fact
that makes it possible to ascertain the presence of the sinus gland in the third
98 ROBERT W. PYLE
stage after hatching. No such innervated structure has been found in the earlier
stages.
In the fourth stage after hatching (Fig. 10) the eye stalk has increased more
in thickness than in length. Consequently, the medulla interna is displaced; the
sinus gland is found on the proximal portion of the medulla externa lateral to the
medulla interna. This brings the sinus gland into closer proximity to the medulla
terminalis. The sinus gland has increased in size with the resultant increase in
the number of nuclei found in it, but the cytoplasm is still lacking the brilliant
acidophilic reaction one might expect. No cell boundaries are visible; the nerve
tract from the medulla terminalis is much more prominent than in the third stage
after hatching.
In the adult (Fig. 11) the sinus gland occupies the same general position as in
the early stages, but the eye stalk has become much more extended so that the
medulla terminalis, interna and externa and lamina ganglionaris are strung out
and occupy a much smaller portion of the inside of the eye stalk than they did
in the early stages. As a result the sinus gland is found in the proximal half of
the eye stalk on the opposite side from the X-organ. It is much more highly-
developed and extended than in the early stages. Situated alongside the medulla
interna and extending to the proximal portions of the medulla externa it sends
large finger-like processes out into the adjacent blood sinus. The nerve fiber
tract extending from the sinus gland to the medulla terminalis is very large;
after the protargol stain of Bodian (1937) one finds that the nerve fibers ramify
among the fibers of the neuropile of the medulla terminalis and branch to all parts
of the sinus gland. The framework of the gland is composed of connective
tissue which stains precisely the same as the other connective tissue found in
the eye stalk. There are no distinct cell boundaries observable in most prepara-
tions, but occasionally one is able to find an isolated cell which has a definite cell
boundary surrounding a large irregular cytoplasmic mass. The nuclei have the
same appearance as those of the early stages; they look more like connective
tissue nuclei than nerve cell nuclei.
As has been pointed out above there is no striking staining reaction in the
sinus gland of the third and fourth stages after hatching. In the adult, however,
there are some interesting phenomena. In the series obtained for this research
the following reactions are discernible: Specimens fixed several days and a few
hours before moulting have the sinus gland filled with irregularily shaped secretory
granules (Fig. 12) which, after Foot's modification of the Masson trichrome stain
and other stains employing acid fuchsin and aniline blue, give a brilliant
acidophilic reaction for the most part although there are a very few granules
which react basophilically. Specimens fixed six hours, 48 hours, one and one
and one-half months after moulting give three characteristic reactions. Some of
the granules are brilliantly acidophilic, some are slightly acidophilic, and a number
are decidedly basophilic (Fig. 13). In specimens fixed four and six months after
moulting the amount of secretory material in the sinus gland is decidedly less
than in those fixed during the summer months at or near the time of moulting
(Fig. 14). In these cases the secretions are for the most part only slightly
acidophilic with an occasional basophilic granule being found. In the specimen
that had not moulted in over a year there was less secretory material in the sinus
gland than was found in those (fixed in the summer months) which had moulted,
SINUS GLAND AND X-ORGAN IN CRUSTACEA 99
but there was more than was found in those specimens fixed in the late fall and
winter. The secretory material was brilliantly acidophilic, slightly acidophilic
and basophilic. There was more basophilic material in this particular specimen
than in any of the others. Examination of the exoskeleton showed that a new
exoskeleton had been laid down underneath the old one which had not, for some
reason, been shed.
C. Cambarus virilis
When the cyclic phenomena were found in the sinus gland of Homarus it
was thought advisable to study the sinus gland of Cambarus virilis in which
Dethier (1942) had previously reported a similar reaction. Accordingly, sections
were made of the eye stalks of specimens fixed just before and just after moulting
as well as of those fixed in late December. The sinus gland of specimens fixed
just before moulting (Fig. 15) was filled with many irregularly-shaped granules
which for the most part gave a brilliant acidophilic reaction, but there were
occasional granules which were basophilic. In those specimens fixed after having
completed moulting (Fig. 16) there was a sharp reduction in the number of
secretion granules present; a few of these were brilliantly acidophilic, but most of
them exhibited varying degrees of a basophilic reaction. In those specimens
fixed late in December (Fig. 17) there were about the same number of granules
as were found in the post-moult specimens, but the majority of these were acido-
philic and only a few basophilic.
DISCUSSION
Dethier (1942) in her account of the sinus gland in Cambarus states that she
has been able to trace it from the first post-embryonic moult, and that it is
apparently functional at that time.7 This is not the case in the two species used
for this investigation; in Homarus it has been impossible to ascertain definitely
its presence until the third stage after hatching, and in Pinnotheres it could not
be detected (with the techniques used) in the egg or first zoeal stages. Cyto-
logically the evidence seems to indicate that the sinus gland in the third and fourth
stages of Homarus is not a functional gland.
It has fairly well established that the color changes in Crustacea are controlled
by hormones which originate in the eye stalk. As Kleinholz (1942) points out
"the glandular tissue is probably the sinus gland, although the X-organ may
also be concerned in this function." The apparent absence of the sinus gland
in the early stages suggests that the X-organ may be functional in this capacity
at this time, but the cytological evidence does not bear this out in Homarus and
Pinnotheres. On the other hand, Cambarus has no X-organ which has the
characteristic concretions of secretory material that are comparable to those
found in Homarus and Pinnotheres. (Welsh, 1941, has found a mass of tissue
on the dorso-lateral side of the medulla terminalis which he suggests may be the
X-organ in Cambarus bartoni.)
Megusar (1912), Abramowitz and Abramowitz (1938, 1940), Brown and
Cunningham (1939), Kleinholz and Bourquin (1941), and Smith (1940) have
7 When the crayfish hatches it is a miniature adult with all appendages etc., and is comparable
to a fifth or sixth stage of Homarus americanus.
100 ROBERT W. PYLE
shown that the removal of both eye stalks from crustaceans hastens the onset of
moulting. Smith showed quantitatively that the removal of both eye stalks
shortened the intermoult period by slightly more than 30 per cent. This probably
indicates that some structure in the eye stalk, possibly the sinus gland, produces
a hormone which has an inhibiting effect upon moulting. Kyer (1942) gives
good evidence that the sinus gland, when active, specifically inhibits moulting
and gastrolith formation. Dethier (1942) in her account of Cambarus suggests
that there is an acidophilic basophilic series which is related to the period of
moulting. In the cases of Homarus and Cambarus the acidophilic reaction before
moulting and the basophilic one after moulting seem to indicate cyclic changes
in the sinus gland which are directly related to the moulting process. Further
evidence of the activity of the sinus gland is exhibited by the reduction in the
amount of secretory material in it; this is most striking in Cambarus, less evident
in Homarus and scarcely detectable in Pinnotheres (this is probably due to the
fact that Pinnotheres passes through several moults in fairly rapid succession).
The explanation of the basophilic and acidophilic reactions in Pinnotheres is
more difficult on the basis of secretory activity. If one had only the normal
animals to consider it might be possible to state that the activities of the sinus
gland in this species passed through a reverse acid-base reaction which were a
direct result of its activity. However, in as much as the sinus gland of the starved
animals, and that in the ones in the process of moulting, both give acidophilic
reactions it may be that the lack of food changes the pH- of the sinus gland from
a normally basic range to an acid range. Since the animal does not feed during
the period of ecdysis this may account in part for the acidophilic reaction of the
sinus gland at this time.
Plankmann (1935) reported that various factors (starvation, etc.) may affect
the rate of moulting. The Pinnotheres that were starved for varying periods of
time were kept at a temperature comparable to that of their normal environment,
on a dark background and in running sea water. There was no increase in the
frequency or number of moults that occurred; it was the sinus gland that showed
the affect of starvation and the X-organ appeared unchanged.
In 'the case of retinal pigment migration Parker (1897) could find no nerve
fibers supplying the distal pigment cells in Palaemonetes. This observation
started the controversy of the interrelationship of the eyes and subsequently
many investigations have been made upon this subject. It has been shown in a
generally satisfactory manner that the sinus gland produces a retinal pigment
hormone (cf. Welsh, 1941). The question is raised as to the mechanism involved
in the early stages where there is no obvious sinus gland to be found. If the sinus
gland is the sole controlling factor it must be assumed that the early stages are
incapable of retinal pigment migration.
Further studies are necessary to give satisfactory answers to the following
points which have not been completely answered in the present study:
1. From precisely what pre-existing tissue is the sinus gland formed?
2. Is the sinus gland a syncytium?
- 3. Is the sinus gland noncellular and merely a storage space or are there cells
which periodically fill with secretory products and break down (e.g. is secretion
holocrine?)?
SINUS GLAND AND X-ORGAN IN CRUSTACEA 101
SUMMARY
1. The histogenesis of the sinus gland and X-organ have been studied and
described for the egg, first zoea and adult stages of Pinnotheres maculatus.
2. The sinus gland is not found in the egg or first zoea, but it is found in all
the adult stages of Pinnotheres.
3. The X-organ is found in the egg and other stages of Pinnotheres.
4. The histogenesis of the sinus gland and X-organ have been studied and
described for the egg, first four stages after hatching and the adult of Homarus
americanus.
5. The sinus gland is not found as a definitely discernible structure in Homarus
until the third stage after hatching.
6. The X-organ is found in all stages of Homarus that have been studied.
7. Evidence is presented for the existence of cyclic secretion phenomena in
the sinus gland of all species studied.
8. There is no evidence of the existence of cyclic secretion phenomena in the
X-organ in any of the species investigated.
A cknowledgments
This research has been carried out with the aid of a fellowship granted by the
Woods Hole Oceanographic Institution. I am indebted to Dr. John H. Welsh,
at whose suggestion this problem has been carried out, for specimens of Cambarus
virilis and the eye stalks of certain stages of the adult Homarus americanus, and
for stimulating discussions of the problem. I am also indebted to Dr. Charles J.
Fish of the Department of Zoology, Rhode Island State College, and the Wickford
Hatcheries, Wickford, Rhode Island, for the eggs, the first four stages after
hatching and the adult eye stalks (before, during and after moulting) of Homarus
americanus. I wish to thank Dr. Gustavus H. Klinck, of the Samaritan Hospital,
Troy, New York, for the use of the microphotographic apparatus.
LITERATURE CITED
ABRAMOWITZ, A. A., AND R. H. ABRAMOWITZ, 1938. On the specificity and related properties of
the crustacean chromatophorotropic hormone. Biol. Bull., 74: 278-296.
ABRAMOWITZ, R. K., AND A. A. ABRAMOWITZ, 1940. Moulting, growth, and survival after eye
stalk removal in Uca pugilator. Biol. Bull., 78: 179-188.
ATKINS, D., 1926. The moulting stages of the pea crab Pinnotheres pisum. Jour. Marine Biol.
Assoc., 14: 475-493.
BODIAN, D., 1937. The staining of paraffin sections of nervous tissue with activated protargol.
Anat. Rec., 69: 153-162.
BROWN, F. A., AND O. CUNNINGHAM, 1939. Influence of the sinus gland of crustaceans on normal
viability and ecdysis. Biol. Bull., 77: 104-114.
DETHIER, F., 1942. Cytological evidences for function in the sinus gland of the crayfish. Thesis,
Harvard University. Unpublished.
FOOT, N. C., 1933. The Masson trichrome staining methods in routine laboratory use. Stain
Tech., 8: 101-110.
HANSTROM, B., 1933. Neue Untersuchungen iiber Sinnesorgane und Nervensystem der Crus-
taceen. II. Zool. Jb. (Abt. Anat.), 56: 367-520.
HANSTROM, B., 1934a. Neue Untersuchungen iiber Sinnesorgane und Nervensystem der Crus-
taceen. III. Zool. Jb. (Abt. Anat.), 58: 101-144.
HANSTROM, B., 1934b. Uber das Organ-X, eine inkretorische Gehirndriise der Crustaceen.
Psychiat. Neural. Bl. Amst., No. 3 en 4: 1-14.
102 ROBERT W. PYLE
HANSTROM, B., 1937. Die Sinusdriise und der hormonal bedingte Farbwechsel der Crustacean.
K. svenska. VetensAkad. Handl. III., 16: 1-99.
HANSTROM, B., 1939. Hormones in invertebrates. Oxford.
KLEINHOLZ, L. H., 1942. Hormones in Crustacea. Bid. Rev., 17: 91-119.
KLEINHOLZ, L. H., AND E. BOURQUIN, 1941. Effects of eye-stalk removal on decapod crustaceans.
Proc. Nat. Acad. Sci., 27: 145-149.
KYER, D. L., 1942. The influence of the sinus glands on gastrolith formation in the crayfish.
Biol. Bull., 82: 68-78.
MEGU§AR, F., 1912. Experimente iiber den Farbwechsel der Crustaceen. Arch. Entw. Mech.
Org., 33: 462-665.
PARKER, G. H., 1897. Photochemical changes in the retinal pigment cells of Palaemonetes, and
their relation to the central nervous system. Bull. Mus. Comp. Zool., 30: 275-300.
PLANKMANN, H., 1935. Beitrage zur Physiologic der Garneelenhautung. Schr. Naturw. Ver.
Schl.-Holst., 21: 195-216.
SCHARRER, B., 1941. Endocrines in invertebrates. Physiol. Rev., 21: 383-409.
SJOGREN, S., 1934. Die Blutdriise und ihre Ausbildung bei den Dekapoden. Zool. Jb. (Abt.
Anat.), 58: 145-170.
SMITH, R. I., 1940. Studies on the effect of eyestalk removal upon young crayfish (Cambarus
clarkii, Girard). Biol. Bull., 79: 145-52.
SrAHi., F., 1938. Uber das Vorkommen vom inkretorischen Organen und Farbwechselhormonen
i,m Kopf einiger Crustaceen. K.fysiogr. Sallsk. Handl. Lund, N. F. 49: 1-20.
WELSH, J. H., 1941. The sinus gland and 24-hour cycles of retinal pigment migration in the
crayfish. Jour. Exp. Zool., 86: 35-49.
NEURULATION IN MECHANICALLY AND CHEMICALLY
INHIBITED AMBLYSTOMA
AGNES SANXAY BURT l
(Department of Zoology, The University of Chicago)
INTRODUCTION
Although the dependence of the medullary plate upon the chorda-mesoderm
has attracted considerable attention from embryologists, the mechanism by which
the plate becomes a neural tube has not been demonstrated.
In amphibians, it has been claimed that pressure exerted by ectoderm and
mesoderm (Giersberg, 1924) or by the liquid confined between those two germ
layers (Rufifini, 1925) is an active factor in neurulation. However, Lehmann
(1926) and Boerema (1929), using different experimental approaches, have de-
monstrated that neurulation in these forms is an autonomous process within the
medullary plate. In echinoderms (Moore and Burt, 1939; Moore, 1941) gastrular
invagination, which in many respects resembles neurulation, has likewise been
shown to be independent of ectodermal pressure.
Mitosis accompanied by a differential increase in cell volume has also been
thought to be a factor in neurulation. Although little or no mitotic activity
during this process was found by Glaser (1914) in Cry ptobranchus alleglieniensis
or by Ruffini (1925) in Triton, the latter worker believes mitosis to be a con-
tributing factor to neurulation in Rana. Derrick (1937) reports that the high
mitotic rate in the sides of the chick medullary plate as compared with the floor
may aid neurulation in that form. In this animal it has also been found that
after the neural tube has closed, incidence of mitosis is higher in the evaginating
optic vesicles than it is in other regions of the brain (Frank, 1925). Hutchinson
(1940), on the other hand, finds that the elongation of the neural tube which
occurs soon after its closure in Amblystoma is not due to cell proliferation.
The hypothesis of Glaser (1914) that neurulation in Cryptobranchus may be
caused by differential water absorption in the medullary plate cells has not been
supported by the data of Brown, Hamburger, and Schmitt (1941) on Amblystoma.
They find no appreciable increase in the water content of the plate during the
critical period as determined by density measurements. Hobson (1941), however,
was able to produce unfolding of partially closed chick neural tubes by dehydrating
them in hypertonic media.
Ruffini (1925) reports that neurulation is aided by autonomous, amoeboid
motion of the medullary plate cells. Boerema (1929) concludes that autonomous
changes in cell shape are the responsible mechanism. It is well established
(Goerttler, 1925; Vogt, 1929; Manchot, 1929, and many others) that extensive
1 This investigation was carried out under the direction of Dr. Paul Weiss. It forms a part
of a thesis on "Chemical Factors in Nerve Development" presented in partial fulfillment of the
requirements for the Ph.D. degree. It has been supported by a grant from the Dr. Wallace C.
and Clara A. Abbott Memorial Fund of the University of Chicago.
103
104 AGNES S. BURT
cell movements take place within the neural ectoderm which result in the elonga-
tion of the structure, but it is not known to what extent these movements are
correlated with the formation of the neural tube.
It was the purpose of the work reported here to compare the cellular changes
taking place in normal embryos during neurulation with those in embryos in which
neurulation had been inhibited by various means in an attempt to find some clue to
the factors responsible.
MATERIALS AND METHODS
Several clutches of eggs of Amblystoma maculatum (Shaw) and of Amblystoma
tigrinum (Green) were used, some of which were obtained near Chicago and some
of which were shipped from Pennsylvania. The eggs were reared at room temper-
ature unless otherwise noted, and care was taken that environmental conditions
should be the same for experimentals and controls in a given series. Stage num-
bers of all specimens refer to Harrison's tables (1918, unpublished).
Most of the embryos were fixed in modified Formol-Zenker, double embedded
in celloidin and paraffin, and sectioned at 6 micra. Some specimens were stained
with Ehrlich's hematoxylin and mucicarmin for the study of cell shape, nuclei,
and pigment granules; others were stained with neutral gentian violet to differ-
entiate yolk and secretion granules. A few embryos were fixed in picric alcohol
and stained with Best's carmine for the determination of glycogen.
MECHANICAL INHIBITION OF NEURULATION
Firstly, mechanical inhibition of neurulation was accomplished as follows:
The medullary plates plus underlying mesoderm were excised from each of two
Amblystoma maculatum embryos in Harrison's Stage 12 and explanted into
Holtfreter's solution. One plate was then placed on top of the other and the two
pieces of tissue weighed down with splinters of cover glass in such a manner that
the plates could not fold up to form a tube. In some cases the plates were
oriented so that the ectoderm of one was in contact with the mesoderm of the
other; in other cases ectoderm was in contact with ectoderm. Six double explants
of this type were studied. A number of intact Amblystoma eggs from the same
clutch from which the membranes had been removed were reared in Holtfreter's
solution, and 10 explanted medullary plates were allowed to develop freely in the
same medium as controls.
The unoperated eggs developed normally except that, in some cases, the
hypertonic medium caused a slight retardation of the head region. By the time
the normal controls had reached Stage 28, the free explants showed distinct signs
of neurulation. When the normal controls were in Stage 31 (Plate I, Fig. 1), the
free explants had prominent neural folds which in some cases had nearly closed to
form a tube (Plate I, Fig. 2). At the same time in the weighted explants, the
medullary cells had elongated and become columnar as in early stages of normal
neurulation, but the flask shape characteristic of later stages was never assumed
and a tube was not formed (Plate I, Fig. 3).
There was no apparent difference in cell shape or intracellular organization
between weighted explants whose ectoderm was in contact with ectoderm and
those whose ectoderm was in contact with mesoderm. Thus it would seem that
NEURULATION IN INHIBITED AMBLYSTOMA
105
J * >••*•«&«
. ^%^^
n ' . .~. it* m A »
PLATE I
FIGURE 1. Neural tube of normal .4. maculatiim embryo. 250 X.
FIGURE 2. Medullary plate from embryo of same chronological age as Figure 1 explanted
into Holtfreter's solution. 250 X.
FIGURE 3. Double explant, same age as Figure 1. ect. = neural ectoderm, mes. = meso-
derm. 250 X.
FIGURE 4. Normal A. tigrinum, Stage 18. 250 X.
FIGURE 5. Ringer-treated embryo, same chronological age as Figure 4. 160 X.
FIGURE 6. LiCl-treated embryo, same chronological age as Figure 4. 160 X.
106
AGNES S. BURT
by Stage 12 the dorso-ventral polarity of the neural plate has already been
established. In both the weighted and free explants the cells were rounder and
shorter than those in the controls, the nuclei were round as compared to the oval
ones in the normal animals, and there was a heavier deposit of pigment granules
around the distal edges of the medullary cells. No other significant differences
were noted.
From these data it was concluded that, while pressure at right angles to the
plane of the medullary plate can inhibit closure of the neural folds, it does not
suppress the initial cell elongation which accompanies that closure.
CHEMICAL INHIBITION OF NEURULATION
Next the developing eggs were subjected to the action of lithium chloride and
of hypertonic salt solutions which, in the proper concentrations, will produce
delayed closing of the neural tube or permanent spina bifida. Three series of
experiments were carried out.
TABLE I
Comparison of development of normal, LiCl- and Ringer-treated Amblystoma.
Figures refer to Harrison s Stages
Normal
controls
M/10
LiCl
Mammalian
Ringer's
Remarks
Series 1
A. tigrinum
20° C.
19
29-30
15
16
16
18-19
34-35
18-19
20-21
neural tube still open in head
region
Series 2
A. maculatum
16-18
13
12
12° C.
19-20
15
12
22-23
18-19
—
died about 140 hours after im-
mersion in salt solution
The first series consisted of three groups of 33 Amblystoma tigrinum eggs which
at the inception of the experiment were in Stage 13. The first group were reared
in well water to serve as normal controls. The second group were reared in
M/10 LiCl solution, the third in mammalian Ringer's. A second series consisted
of three groups of 17 A. maculatum eggs which at the beginning of the experiment
were in Stage 12 b. As with the tigrinum eggs, one group was reared in well
water, one in M/10 LiCl, and one in mammalian Ringer's. However, the
maculatum eggs, instead of being kept at room temperature, were placed on a
water table with a practically constant temperature of 12° C.
The nervous systems of the treated animals in both series diverged consider-
ably from the mean of normal development. In general, the head region was
more retarded than the spinal cord . The approximate degree of maturity attained
by the experimentals in comparison with the controls is shown in Table I. In
staging the treated animals, external appearance was the criterion used.
It should be noted that the difference between the normal and lithium-treated
embryos is greater at 20° C. than at 12° C. (this confirms the work of Hall (1942)
NEURULATION IN INHIBITED AMBLYSTOMA
107
on Rana pipiens} but that low temperatures apparently augment the effect of
Ringer's solution.
The third series consisted of three groups of 17 A. tigrinum eggs which were in
Stage 1 1 b — 12 a at the beginning of the experiment. One group served as normal
controls, one group was immersed in M/20 LiCl for 24 hours, after which develop-
ment was allowed to continue in well water, and the third group was similarly
treated with M/20 XaCl. NaCl treatment had no perceptible effect on the rate
or type of development, while the equimolar LiCl solution retarded the embryos
considerably. This series of eggs was fixed in picric alcohol for a rough determi-
nation of glycogen content.
Effects of chemical inhibition on mitotic rate
The effects of chemical inhibition were best seen in the first series of eggs as
the Ringer-treated eggs did not develop at all in the second series. One-third of
TABLE II
Mitotic rate in the medullary plate of A. tigrinum. Stage numbers not in parenthesis refer to
normal controls; those in parenthesis refer to inhibited animals of the same
chronological age as the normal controls
Stage
Cells
counted
Mitoses
seen
Mitotic
index
Normal controls
18
30
35
1461
776
1037
39
29
42
2.67%
3.73%
4.05%
Lithium chloride-
treated
18(15)
30(16)
35(18)
1970
1109
693
19
12
3
0.96%
1.0895
0.43%
Ringer's treated
18(16)
30(18)
35(20)
2276
760
1074
28
18
30
1.23%
2.37%
2.79%
the embryos were fixed and sectioned when the normal controls were in Stage 18
(at which time the normal germs had open medullary plates with well raised
neural folds), one-third when the controls were in Stage 30, and the remainder
when the controls were in Stage 35, by which time the lithium embryos were in
approximately the same stage of development as the controls at Stage 18 as far as
external appearance was concerned, and the Ringer-treated germs were slightly
more mature. The effect of this inhibition on the mitotic rate in the medullary
plate is summarized in Table II.
From this it is apparent first, that there is mitosis in the neural plate of A.
tigrinum during neurulation; secondly, that the mitotic rate rises in the normal
animal after the neural tube is closed; thirdly, that Ringer's solution depresses
the mitotic rate in comparison with normal embryos of the same chronological
age, but that the mitotic rate in Ringer-treated animals is comparable to that in
normals of the same stage of development, and fourthly, that LiCl causes both a
relative and an absolute decrease in the mitotic rate of the neural tube.
108 AGNES S. BURT
Effects of chemical inhibition on cell shape
The effects of inhibition on the cellular morphology of the neural tube were
extreme. When the normal controls were in Stage 18 (Plate I, Fig. 4), the Ringer-
treated animals showed a slight evagination of the floor of the medullary plate
(Plate I, Fig. 5), and lithium-treated embryos a very marked evagination (Plate I,
Fig. 6).
By the time the normal controls were in Stage 30 (Plate II, Fig. 7), ectoderm
had begun to grow over the edges of the plate in the Ringer-treated germs and a
slight invagination of the plate was present (Plate II, Fig. 8). When the con-
trols were in Stage 35 (Plate II, Fig. 10) and the Ringer-treated embryos in what
corresponded to Stage 20 in the normal animals, the invagination was fairly deep
in the treated germs and the edges of the plate were raised, although they were
not bent over as normal neural folds are at that time (Plate II, Fig. 11).
In the LiCl-treated germs, on the other hand, when the controls were in
Stage 30, a flat plate was present (Plate II, Fig. 9). When the controls had
reached Stage 35 and the lithium-treated animals were in Stage 18 as far as ex-
ternal appearance was concerned, the neural plate was still flat, but a few flask-
shaped cells had appeared at the edges as in the first stages of normal neurulation.
Many of the medullary plate cells in these embryos, particularly in the head
region, became round and sloughed off into the space above the plate (Plate II,
Fig. 12). Child (1941) reports a similar dissociation of the endodermal plate in
the starfish, Pateria, when exposed to the action of lithium chloride.
The changes in shape occurring in both the normal and treated embryos
naturally correspond to the changes in the shape of the plate as a whole. These
changes may be summarized by saying that both Ringer and LiCl treatment
produce, first, a more or less evaginated medullary plate and then a flat or slightly
invaginated plate which may, according to the concentration of the chemicals
used, proceed to form a tube in places or to be overgrown by ectoderm, and that
no traces of a neural keel as described by Baker (1927) were seen in the treated
embryos in the series studied.
Effects of chemical inhibition on nuclear size
Much importance has been attached to changes in cell and nuclear size during
neurulation since Glaser (1914) found that, in Cryptobranchus, the volume of the
neural plate increased during the course of neurulation and believed that this
indicated an increasing water content of the neural plate. He also inferred that
increased hydration occurs during gastrulation in echinoderms because of reported
increases in nuclear size during that process. As Brown, Hamburger, and Schmitt
(1941) found no indications of increased hydration in density measurements on
Amblystoma, an effort was made to throw more light on the problem by measuring
the nuclear axes of 100 medullary plate cells in both normals and experimental
in each of three stages. As these nuclei are not perfect spheres and as their
orientation varies somewhat within the plate, these measurements cannot be used
to calculate nuclear volume. However, any large changes in nuclear volume
should be revealed by this method. Indices of nuclear area and shape were also
calculated. These data are summarized in Table III.
NEURULATION IN INHIBITED AMBLYSTOMA
109
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A* - •«-•«» .%-AV * ^P
jpir-jStf- * *#•«&
»W.;£r * ^i&K?
fc;#?fiE#$ ^i^SS
,.,v» » >s» •. -V- ™
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PLATE II
FIGURE 7. Normal .4. tigrinum embryo, Stage 30. 250 X.
FIGURE 8. Ringer-treated embryo, same chronological age as Figure 7. ect. = ectoderm
growing over medullary plate. 160 X.
FIGURE 9. LiCl-treated embryo, same chronological age as Figure 7. 160 X.
FIGURE 10. Normal A. tigrinum embryo, Stage 35. 250 X.
FIGURE 11. Ringer-treated embryo, same chronological age as Figure 10. 160 X.
FIGURE 12. LiCl-treated embryo, same chronological age as Figure 10. Note round cells
sloughed off from plate. 250 X.
110
AGNES S. BURT
No statistically significant differences in nuclear axes, area, or shape were re-
vealed by this analysis between normal and treated nuclei because of the large
standard deviations involved. However, it should be noted that the index of
shape (A/B) increased consistently in the normal germs whereas it remained
practically constant or decreased slightly in the chemically treated germs. This
lack of nuclear elongation seems to be correlated with the failure of cell elongation
which was also observed in these cases. While no conclusions can be drawn from
these findings as to cellular hydration, there is no change of nuclear size during
folding of the neural plate.
Effects of chemical inhibition on cellular inclusions
A. Yolk granules. In normal A. tigrinum and A. maculatum embryos, yolk
begins to be utilized in the neural tube, beginning in the head region, about
TABLE III
Greatest nuclear length (A) and diameter (B) of 100 medullary plate cells. Stage numbers not in
parenthesis refer to normal controls; those in parenthesis refer to treated animals.
All measurements are in ocular micrometer units
Stage
Nuclear measurements
Indices
Length (A)
Diameter (B)
Area (AB)
Shape (A/B)
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
Normal controls
18
30
35
15.00
14.60
16.80
2.22
2.76
2.83
8.84
8.42
8.86
1.58
1.56
1.52
129.4
119.2
146.6
34.8
30.6
36.8
1.72
1.80
1.93
0.35
0.51
0.48
Ringer-treated
18(16)
30(18)
35(20)
13.55
14.33
13.27
2.41
2.24
2.18
8.66
9.03
9.42
1.56
1.71
1.56
115.9
126.7
121.7
33.4
38.8
32.2
1.59
1.58
1.42
0.38
0.36
0.32
LiCl-treated
18(15)
30(16)
35(18)
14.79
14.63
13.96
2.21
2.22
2.03
9.51
10.79
10.02
1.46
1.56
1.17
139.7
157.3
138.7
34.4
38.0
32.0
1.45
1.26
1.38
0.27
0.25
0.20
Stage 18. By Stage 35, yolk has practically disappeared from the brain, and
only a sparse scattering of granules remains around the lumen of the spinal cord.
Bragg (1939), who has studied the utilization of yolk in a number of other am-
phibian genera, reports that in his animals it did not begin until after the closure
of the neural tube.
Lithium chloride and Ringer's solution both seem to retard the disappearance
of yolk as well as the closure of the neural tube, as the medullary plates of the
treated animals were packed with yolk granules throughout the period of observa-
tion whereas, in the normal controls, the amount significantly diminished. It is
doubtful if this is causally related to the process of neurulation, however, because
(1) yolk disappears very late in this process and (2), as Morgan (1906) has shown,
eggs of Bufo variabilis centrifuged so that all granules are throwrn out of portions
of the head in the resultant embryos will develop closed neural tubes.
NEURULATION IN INHIBITED AMBLYSTOMA 111
B. Glycogen. In the A. tigrinum scries, no perceptible change in the glycogen
content of the nervous system was noted between Stage 18, at which time the
neural tube is open over its full length, and Stage 30, when the entire tube is closed
and morphogenesis of the brain is well under way. All the neural cells contained
much glycogen, no significant differences being noted among the various regions
of the nervous system.
Treatment with M/20 NaCl did not affect glycogen distribution (as was to be
expected since no morphological changes were observed) nor did treatment with
M/20 LiCl. Thus, although no histological method is exact enough to reveal very
small changes in glycogen content, it would appear that in A. tigrinum neurulation
is not accompanied by significant utilization of this material.
C. Pigment granules. Early in the normal process of neurulation in Am-
blystoma, as reported by Ruffini (1925) and Lehmann (1926) for other urodeles,
there is a marked accumulation of pigment, especially in creases formed by the
medullary folds. From the pigment layer at the distal ends of the cells, rowrs of
pigment granules extend along the cell boundaries (see Plate I, Fig. 1). When
neurulation is completed, there is a layer of pigment granules along both surfaces
of the neural tube and many granules along the cell boundaries and within the cell
bodies.
The chief difference noted in the treated animals was that the concentration
of pigment near the outer surface of the plate cells occurred irregularly and only
in those cells where shrinkage of the inner surface took place (see Plate I, Figs. 5
and 6; Plate II, Figs. 8 and 12). In the lithium-treated specimens, as soon as
degeneration of the plate commenced, many granules escaped into the free space
above the plate, and all of the sloughed-off cells were packed with pigment. These
granules, like yolk, however, appear to be a passive factor in neurulation and are
of value only as an indicator of the results of active processes which change cell
shape.
D. Secretion granules. As shown by Studnicka (1900) and \Veiss (1934),
secretion occurs in the embryonic ependyma. On the chance that secretion might
be involved in the process of neurulation, normal and chemically inhibited A.
tigrinum, A. maculatum, and Rana pipiens germs and normal chick embryos which
had been stained with neutral gentian violet were examined for secretion granules.
None were found, either in the normal embryos or in the treated amphibians.
This does not necessarily indicate that unformed secretion does not occur; in fact,
the presence of liquid within the lumen of the neural tube is evidence that secretion
of some sort does take place very early in normal development. Because of the
difficulty of demonstrating secretion antecedents, however, the problem requires
study by more refined techniques.
Effects of chemical treatment on cellular movements
The most accurate method of following cell movements in embryonic develop-
ment is by vital staining, a procedure not used in this investigation. However,
some indication of those movements was obtained by counting the number of cells
in the neural plate in every fifth section of the trunk region of embryos (exclusive
of brain and tail) and averaging the results. It is very hard to obtain strictly
comparable results by this method because, as Manchot (1929) has shown, in the
normal development of urodele embryos, the anterior two-thirds of the neural
112
AGNES S. BURT
plate becomes brain while the posterior one-third elongates to form the spinal
cord. A rough comparison between the normal and chemically treated neural
plates at various stages of development is presented in Graph 1.
As this graph was constructed from data on only nine animals, not too much
significance can be attached to it. However, it would seem that in normal ani-
mals between Stages 18 and 35 mitosis and stretching of the neural plate keep pace
with one another so that the average number of cells per cross section remains
225
200
o
U'75
X
UJI50
(S)
125
LJ
O
MOO
75
\
\
\
\
\
\
8
30
35
STAGE
GRAPH 1. Average numbers of cells present in every fifth section of the medullary plate of
normal and chemically treated A. tigrinum embryos of the same chronological age. Abscissae
are stage numbers of normal controls. Solid line used for normal controls, dashes for lithium
embryos, dots for Ringer germs.
approximately constant and that, although LiCl and Ringer's solution do inhibit
the stretching process just as they inhibit neurulation and mitosis, elongation of
the neural plate continues under their influence.2
- In Glaser's work on Cryptobranchus (1914), he used the average number of nuclei present
per cross-section to test whether or not cell division was occurring. Because the number of
nuclei remained approximately constant, as it does in normal Amblystoma during slightly later
stages, he concluded that there was little or no mitosis during neurulation. It would be interesting
to re-examine his material to see if elongation of the medullary plate played any role in that
constancy.
NEURULATION IN INHIBITED AMBLYSTOMA 113
DISCUSSION
In discussing neurulation, it must be borne in mind that the folding of the
neural plate is a very complex process involving not only changes in cell size and
proportions, but physical and biochemical changes which have, as yet, been little
studied. From the foregoing analysis, it can be concluded that certain factors
are not involved in the more obvious phases of folding. Thus not only does
neurulation occur, as has been previously reported by many workers, in the ab-
sence of normal mechanical pressures, but the characteristic preliminary cell
elongation takes place when pressure is exerted at right angles to the usual direc-
tion of cell movement.
Neurulation seems to be independent of nuclear area, and if, as has been sug-
gested, the latter be accepted as an index of cell hydration, also independent of
hydration. For, although treatment with Ringer's solution and LiCl had no
significant effect on nuclear area, it did inhibit folding of the medullary plate.
On the other hand, nuclear elongation, which accompanies cell elongation in the
normal plate, does not occur when folding is inhibited.
Judging by the data on average number of cells per section, the elongation of
the medullary plate which normally takes place during and after neurulation is not
necessarily correlated with the closure of the neural tube, because, although both
LiCl and Ringer's solution do retard the stretching process somewhat, it continues
even when neural folds do not form.
Mitosis seems to be either directly instrumental in neurulation or at least under
control of the mechanism of neurulation. Thus in the LiCl-treated embryos, in
which the mitotic rate fell to a very low value during the experiment, no folds
appeared, while the elevation of the sides of the neural plate in both the normal
and the Ringer-treated specimens was accompanied by active cell division.
Cell elongation and wedging are unavoidably correlated with embryonic
folding, and, as Boerema (1929) and others have pointed out, such changes are
theoretically quite sufficient to cause neurulation. As demonstrated by Brown,
Hamburger, and Schmitt (1941) differential water absorption cannot account for
such changes in Amblystoma. These workers and Schmitt (1941) independently
have suggested that molecular interactions and desolvations in the cell surface
may exert the forces necessary to cause cell elongation. Weiss (unpublished) has
suggested further that the concentration of pigment granules which occurs in the
normal folding plate indicates a contraction of the cell cortex at the free surface.
Although Hobson (1941) has not succeeded in demonstrating any systematic
changes in the ultrastructure of the chick neural plate during folding by polariscopic
analysis, a more intensive investigation of such changes during neurulation ap-
pears to be the most promising method of approach to the problem.
An interesting point which emerges in a comparison of the LiCl and Ringer-
treated germs is that the effects of the two agents on neurulation seem to be pro-
duced by different means. Thus lithium is less effective at low temperatures,
while hypertonic salt solutions are more effective. Further, lithium salts inhibit
neurulation at much lower concentrations than do those present in Ringer's solu-
tion. Hall (1942) has evidence that lithium is a toxic agent acting on the chorda-
mesoderm rather than on the responding ectoderm. The fact that Ringer's solu-
tion is a more effective inhibitor at low than at high temperatures would suggest
that it acts on the physical consistency of the embryo rather than on chemical
114 AGNES S. BURT
processes, perhaps by stiffening the neural plate so that folding is impeded — a
suggestion which Giersberg (1924) has previously offered to explain the action of
sucrose and sodium acetate on neurulation.
Finally, it should be noted that the data presented here are by no means con-
clusive in themselves; they are offered merely in an effort to shed light on a few
phases of a very complicated problem.
SUMMARY
1. Mechanical pressure exerted at right angles to the plane of explanted
medullary plates has been found to suppress neurulation in A mblystoma maculatum,
but not the preliminary cellular elongation which is normally involved in that
process. This elongation takes place irrespective of whether the medullary plate
is in contact with ectoderm or with mesoderm on the normally free surface.
2. In Amblystoma tigrinum and A. maculatum neurulation is accompanied by
mitosis, the mitotic rate rising after the neural tube has closed. Treatment with
mammalian Ringer's solution at room temperature decreases the mitotic rate to
about the same degree as it inhibits normal development; treatment with M/10
LiCl decreases the mitotic rate both relatively and absolutely.
3. No statistically significant difference was found in average nuclear area be-
tween normal and treated medullary plates. In normal germs, the nuclei
elongate during neurulation, whereas in the treated germs they did not.
4. Glycogen and yolk begin to disappear from the normal neural tube about
Stage 18. Neurulation-inhibiting chemicals retard the utilization of these
substances.
5. Pigment granules appear to be passive factors in neurulation indicative of
contraction at free cell surfaces.
6. No evidence of formed secretion from the neural plate was found.
7. Although inhibiting chemicals decrease the rate of elongation of the
medullary plate, stretching continues even when neural folds fail to form.
8. The inhibiting action of LiCl is less effective at low temperatures, that of
Ringer's is augmented.
9. It is concluded that neurulation in Amblystoma is autonomous to the
medullary plate and may be aided by mitotic activity; changes in nuclear area
(which may be indicative of cell hydration), intracellular inclusions, and longi-
tudinal cell movements are not instrumental in the process.
LITERATURE CITED
BAKER, R. C., 1927. The early development of the ventral part of the neural plate of
Amblystoma. Jour. Comp. Neur., 44: 1-27.
BOEREMA, I., 1929. Die Dynamic des Medullarrohrschlusses. Arch.f. Entwmech., 115: 601-615.
BRAGG, A. N., 1939. Observations upon amphibian deutoplasm and its relation to embryonic
and early larval development. Biol. Bull., 77: 268-284.
BROWN, M. G., V. HAMBURGER AND F. O. SCHMITT, 1941. Density studies on amphibian embryos
with special reference to the mechanism of organizer action. Jour. Exp. Zool., 88:
353-372
CHILD, C. M., 1941. Patterns and problems of development. University of Chicago Press.
DERRICK, G. E., 1937. An analysis of the early development of the chick by means of the mitotic
index. Jour. Morph., 61: 257-284.
FRANK, G. M., 1925. Uber Gesetzmassigkeiten in der Mitosenverteilung in den Gehirnblasen in
Zusammenhange mit Formbildungsprozessen. Arch.f. Entwmech., 104: 262-272.
NEURULATION IN INHIBITED AMBLYSTOMA 115
GIERSBERG, H., 1924. Beitrage zur Entwicklungsphysiologie der Amphibien. II. Neurulation
bei Rana und Triton. Arch.f. Entwmech., 103: 387-424.
GLASER, O. C., 1914. On the mechanism of the morphological differentiation in the nervous
system. I. The transformation of a neural plate into a neural tube. Anal. Rec., 8:
525-551.
GOERTTLER, K., 1925. Die Formbildung der Medullaranlage bei Urodelen. Arch.f. Entwmech.,
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HALL, T. S., 1942. The mode of action of lithium salts in amphibian development. Jour. Exp.
Zool., 89: 1-36.
HOBSON, L. B., 1941. On the infrastructure of the neural plate and tube of the early chick embryo,
with notes on the effects of dehydration. Jour. Exp. Zool., 88: 107-134.
HUTCHINSON, C., 1940. A study of medullary plate formation in Amblystoma punctatum.
Anal. Rec., 78 (Suppl.): 56.
LEHMANN, F. E., 1926. Entwicklungsstorungen in der Medullaranlage von Triton, erzeugt
durch Unterlagerungsdefekte. Arch.f. Entwmech., 108: 243-282.
MANCHOT, E., 1929. Abgrenzung des Augenmaterials und anderer Teilbezirke in der Medullar-
platte; die Teilbewegungen wahrend der Auffaltung (Farbmarkierungsversuche an
Keimen von Urodelen). Arch.f. Entwmech., 116: 689-708.
MOORE, A. R., 1941. On the mechanics of gastrulation in Dendraster eccentricus. Jour. Exp.
Zool., 87: 101-111.
MOORE, A. R., AND A. S. BURT, 1939. On the locus and nature of the forces causing gastrulation
in the embryos of Dendraster eccentricus. Jour. Exp. Zool., 82: 159-171.
MORGAN, T. H., 1906. The influence of a strong centrifugal force on the frog's egg. Arch. f.
Entwmech., 22: 553-563.
RUFFINI, A., 1925. Fisiogenia. La biodinamica dello sviluppo ed i fondamentali problemi
morfologici dell'embriologia generate. F. Vallardi, Milan.
SCHMITT, F. O., 1941. Some protein patterns in cells. Growth, 5 (Suppl.): 1-20.
STUDNICKA, F. K., 1900. Untersuchungen iiber den Bau des Ependyms der nervosen Central-
organe. Anal. Hefte, 15: 303-431.
VOGT, W., 1929. Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. II. Teil.
Gastrulation und Mesodermbildung bei Urodelen und Anuren. Arch. f. Entwmech.,
120: 384-706.
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Anal. Rec.. 58: 299-302.
ANALYSIS OF POPULATION DEVELOPMENT IN DAPHNIA AT
DIFFERENT TEMPERATURES
DAVID M. PRATT
(From the Biological Laboratories, Harvard University, Cambridge)
INTRODUCTION
The purpose of this study was to analyze the development of Daphnia popula-
tions under controlled conditions in which temperature was the chief variant. It
was proposed to investigate not only the effect of temperature upon the rate of
increase, but also its influence upon subsequent changes in the numerical strength
of the population. The original intention was to measure the effect of tempera-
ture by comparing the equilibrium values, i.e. asymptotes, attained by populations
at different temperatures, and through further experiments to identify the proc-
esses by which temperature might act to bring about the observed differences.
However, the type of population growth curve obtained precludes the comparison
of asymptotes and necessitates a brief historical sketch of population studies in
general and the curves developed from them, in addition to a review of previous
research upon the effects of temperature.
Since the animal chosen for the investigation is a planktonic form, the analysis
of the relation of temperature to the development of a population acquires addi-
tional interest from an old controversy. It has long been held that the polar
regions support a more abundant, if a less diversified, fauna and flora than do the
tropics. This contention has been stressed especially in connection with the
latitudinal distribution of plankton, and a number of theories have been advanced
relating temperature, directly or indirectly, to the density of planktonic popula-
tions. The relation of the present study to this problem and other possible
applications will be dealt with in the discussion.
HISTORICAL BACKGROUND
The logistic equation and its sigmoid curve, rediscovered by Pearl and Reed
(1920), have been applied to the study of human populations (Pearl, 1925) and
experimental populations of a variety of organisms, including yeast cells (Clark,
1922; Richards, 1928), diatoms (Ketchum and Redfield, 1938), infusorians
(Robertson, 1921, 1923), and flour beetles (Chapman, 1928; Holdaway, 1932).
Indeed no population study under controlled environmental conditions has demon-
strated any other type of population growth curve. It should be noted, however,
that the interest of investigators of experimental population development has been
focussed almost exclusively on the early parts of the growth curve, with very little
regard for the important part of the history which follows the initial period of
increase. Although the definition of a logistic curve requires an upper asymptote,
some workers have followed the development of their experimental populations
only to the point where they first approach an apparent maximum size, and in
116
TEMPERATURE AND DAPHNIA POPULATIONS 117
presenting their results have termed their curves logistic. While it is true that
in several studies (e.g. Pearl with Drosophila, 1925; Cause with yeast, 1932;
Chapman with Tribolium, 1928) the population has maintained an upper
asymptote for a period long compared to the "growth" period, it does not follow
that populations of other species or under different circumstances would yield
similar results. The tacit assumption that an asymptote can be calculated from
the maximum size reached by a population, without experimental evidence that a
state of relative equilibrium has been attained, is entirely gratuitous. It may
well be that the failure to demonstrate, hitherto, a type of population growth curve
that is not logistic after the initial period of increase, has been due in part to this
fallacious assumption.
The rather extensive literature on population studies yields but a meager
amount of information concerning the effects of temperature. The yeast
Saccharomyces cerevisiae has been the subject of two investigations involving tem-
perature. Richards (1928a) found that the rate of multiplication increases with
temperature between 4° and 30° C.; above this range it decreases. In a more
thorough analysis of the effects of temperature, Cause (1932) followed the de-
velopment of yeast populations to their asymptotes and discovered that in a tem-
perature range of 5.7° to 41.0° C. the relation between the size of the asymptotic
population and the temperature can be expressed by a bell-shaped curve with the
mode at about 24° C. In the same paper Cause reported that populations of
Drosophila held at 29° C. attain an asymptote of 310, whereas at 30° the asymptote
is only 146.
Terao and Tanaka (1928, 1928a, 1928b, 1930) attempted the study of the
influence of temperature on population development in Moina macrocopa, but
followed their population growth curves only to apparent maxima, and based
their conclusions on the calculated values of undemonstrated asymptotes.
MATERIALS AND METHODS
Daphnia magna would appear to be ideal material for population studies be-
cause of its size, high reproductive capacity, and parthenogenesis, which makes it
easy to obtain genetically identical material. Daphnia is less easily provoked to
the production of males and sexual females than other cladoceran genera (e.g.
Moina), so that in a crowded population a very high percentage of the individuals
are potential producers, and there is no problem of a proper balance of sexes. In
these experiments the sex ratio was noted at irregular intervals, and at no time did
the males constitute more than 10 per cent of the population. Since cladocerans
pass their early stages in the maternal brood pouch whence they are released in
active state, there are no stages (such as eggs) so small as to require special pre-
cautions against loss during transfer of the population to fresh medium.
The populations developed in 50 cc. of filtered pond water from the Middlesex
Fells, in open, wide mouthed glass bottles whose water-surface area was 10.9 cm.2
The seeding of each bottle was two animals (parthenogenetic females) that had
been released from the brood pouch within 24 hours. Each population was
counted every two days, at which time the dead were removed, their number
noted, and the water changed. This was done by pouring the contents of each
bottle into a fingerbowl, whence the animals were transferred with a pipette to a
118 DAVID M. PRATT
second fingerbowl containing about 50 cc. of fresh pond water at the same temper-
ature, and thence into a clean bottle which was finally filled up to the 50 cc. mark
with fresh pond water. By this rinsing process the small amount of used water
carried over in the pipette was greatly diluted. Thus "conditioning" of the
medium by the metabolic activities of the animals was never allowed to proceed
for more than two days.
The only food used was Chlorella pyrenoidosa, a unicellular green alga that will
grow in a thick suspension when properly cultured (in Detmer's Solution, exposed
to neon light, with carbon dioxide bubbling through the medium). It was found
necessary to culture the Chlorella under sterile conditions to prevent the develop-
ment of a concentrated bacterial flora in the culture flasks. In previous experi-
ments contamination of the Daphnia medium from this source had occasionally
been sufficiently severe to injure the animals. The quantity of food given each
population was not measured by any absolute standard. In each case it was
roughly calculated, by previous experience alone, to exceed the requirements of
the particular population. This method proved entirely satisfactory, for the
medium always had a distinct greenish tinge. It was also demonstrated by
simple experiments that when the concentration of the Chlorella was half as great
or several times as great as the concentration that normally would have been used
under the given conditions, the longevity and reproductive rate of the animals
were not appreciably affected. Therefore neither a lack nor an excess of food was
ever a limiting factor in the growth of the populations.
The temperatures chosen for comparison were 12°, 18° and 25° C., covering a
considerable portion of the range (8° to 28° C.) demonstrated suitable for the life
and reproduction of Daphnia magna (MacArthur and Baillie, 1929). At 12°,
however, populations persisted for only a few weeks of faltering growth and
rapidly dwindled to extinction. Under the ecological conditions that obtained,
apparently the metabolic rate was not high enough to insure the reproductive and
survival rates requisite for population growth and maintenance. In consequence
the lowest temperature was abandoned and the work was limited to two tempera-
tures, 18° and 25° C. The populations were maintained at these temperatures
(plus or minus 1° C.) by keeping them in incubators in a cold-room.
The culture bottles were placed in daylight from a north window. However,
all the populations received approximately the same amount of light. Aside from
this, no attempt was made to control light conditions, which varied from day to
day and from season to season.
The only environmental agencies that suggest themselves as possible limiting
factors in the growth of populations of such an animal as Daphnia are: 1. exhaus-
tion of the food supply and 2. conditioning of the medium by the accumulation of
metabolites and/or depletion of the dissolved oxygen. Since the former was never
operative in these experiments, any limitation in the increase in numbers must
have been the expression of some form of conditioning of the medium, although
that process was never continuously sustained for more than two days. In an
attempt to ascertain the nature of this conditioning, the concentrations of hydro-
gen ion, dissolved oxygen and free carbon dioxide in the culture medium were
determined at various densities of population.
The pH, as determined with a Hellige Comparator, never left the range
6.9-7.1, and even within these narrow limits it was not correlated with the popula-
TEMPERATURE AND DAPHNIA POPULATIONS 119
tion density nor with the length of time that animals had been living in the water.
It can be said with a fair degree of certainty that the hydrogen ion concentration
never exerted an important influence upon population growth.
The concentration of free carbon dioxide was determined by a titration method
reported in a publication of the American Public Health Association (1936).
Thus determined, the amount in unused pond water at 18° or at 25° was imper-
ceptible. At the end of a two-day period, crowded populations at 18° had raised
the concentration to an average of 4.97 p. p.m.; at 25°, to 5.66 p. p.m.
Winkler Method determinations of the dissolved oxygen concentrations of
fresh pond water and water conditioned by large populations for two days yielded
the following results: 1. At 18°, unused water 8.38 p. p.m.; after two days' con-
ditioning 6.59 p. p.m. and 2. at 25°, unused water 7.57 p. p.m.; after two days
conditioning 4.81 p. p.m. Each of these figures is the average of ten determinations.
There is no evidence available at the present time as to whether or not these
slight changes in carbon dioxide and oxygen are sufficient to account for the
limitation in population growth. It is also possible that the limiting factor be
some metabolite such as that postulated by Brown and Banta (1932) for male
production.
COURSE AND ANALYSIS OF POPULATION DEVELOPMENT
The problem of determining the influence of temperature upon population de-
velopment resolves itself into two phases: 1. a descriptive study of the observable
effects of temperature upon the form and dimensions of the population curve, and
2. an analysis of the processes through which the difference in temperature brings
about the observed results. The present section is confined to the presentation
of the factual data on the history of populations at 18° and 25° and the discussion
of these growth curves. The analysis of the influence of temperature will be dealt
with in later sections.
A . Observations at 25° and at 18°.
At 25°, four series of populations were started on different dates in January
and February 1942. The histories of these 21 populations were recorded either
until their natural extinction or until September 13, 1942, when all remaining
populations were discontinued. Graph I presents the observations on a typical
series, and reveals that the 25° population curve is characterized by violent and
fairly regular oscillation. Instead of terminating in an upper asymptote, the first
period of increase results in a pronounced peak, after which the curve drops almost
to the baseline, then repeats the cycle. Typically there is no asymptote.
The majority of the populations became extinct before the experiment was
terminated. Those that survived until the 234th day, when observations ceased,
described, commonly, four major oscillations in numbers. The maximal size
attained was a population of 126 animals.
At 18°, three series of populations, started on different dates in late March
1942, were followed until September 13, 1942, when the experiment was termi-
nated. None of these 16 populations became extinct in the 174 days of observa-
tion. Graph II, presenting the histories of a typical series, shows that each curve
described a prominent peak, followed by a gradual decrease and virtual stabiliza-
120
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tion or continued oscillations of relatively minor amplitude. The greatest
maximum achieved was 241 animals in the 50 cc. environment, and after the
major increase, a density of more than 100 animals was permanently maintained
in all the populations. While the course of development at 18° is oscillatory, it
differs from that at 25° in various points that will be examined later.
Preliminary experiments in which the volume of the medium used had been
100 cc. rather than 50 cc. yielded similar results with regard to the relative shapes
of the curves, and population maxima of 381 individuals at 18° C. and 296 at
23° to 24° C.
B. Analysis of oscillation.
Since oscillation is especially pronounced at 25°, the discussion of this phe-
nomenon will be illustrated with the data from that temperature. The analysis
of fluctuations in the size of a population is essentially the study of changes in the
ratio of births to deaths. Whereas the fundamental feature of an asymptotic
population is that at some point birth rate and death rate become equal and there-
after remain constant, in an oscillatory population the curves describing birth
rate and death rate repeatedly cross each other and never remain equal. The
present fluctuations might be due to oscillation of the birth rate about a constant
death rate, or to the converse, or to differential changes in both rates. In order
to establish the cause of the fluctuations, it is therefore necessary to ascertain by
which of these three methods the ratio of births to deaths varies.
Daily tabulation of births and deaths revealed that the oscillations observed
in the 25° populations resulted from changes in both the number of births and the
number of deaths. Periods of increase in population size were marked by a com-
bination of high reproductive activity and low mortality ; decreases were caused by
increased mortality coupled with negligible reproduction. This mechanism of
oscillation, in terms of the changing births/deaths ratio, is illustrated in Graph III
which depicts part of the history of a representative 25° population (No. 2 in
Graph I), with curves showing the numbers of births and deaths for each day of
population census.
The history of one complete cycle will illustrate the reasons for these changes
in the ratio of births to deaths. At the outset of an upward swing, the population
consists in a few adults. Having lived the greater part, if not the whole, of their
lives under favorable environmental conditions as regards crowding, these indi-
viduals exhibit a high reproductive rate. The growing population is composed
of a few (two to 10 or 15) rapidly reproducing adults and their much smaller
offspring. Graph III shows that the increasing population density begins to exert
its harmful effects upon the reproductive rate before it affects the death rate, as
it does in growing populations of Drosophila (Pearl, 1927). Thus the reproductive
activity of the few adults in the population gradually dwindles, and the population
reaches the maximum. The crucial and distinctive crossing of the birth and
death curves at about this point is ascribed to two factors: 1. the extent of
biological conditioning that occurs in the 48 hours between changes of the medium
is presumably greater at this density than ever before, and 2. the cumulative
adverse effects of crowding upon animals that have lived the greater part of their
lives at high population densities begin to manifest themselves. The effect of
these factors is sudden and severe: the death rate soars and reproduction is greatly
TEMPERATURE AND DAPHNIA POPULATIONS
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reduced. The rapidly growing population has "overshot" the density which it
might theoretically be just capable of sustaining.
That the population overshoots the density of potential equilibrium does not
explain why it drops dangerously close to extinction before recovering itself. At
a point, for example, halfway in its descent it might be expected to rally its forces
and start up again, since this same intermediate density on the upswing had been
correlated with a high reproductive rate and low mortality.
In seeking the reason for the difference in performance of the two populations
of equal density, it should be borne in mind that the composition of the waning
population at any given point differs significantly from that of the waxing popula-
tion of the same numerical strength. For example, the average size of the indi-
viduals, and hence the total biomass, of the waning population is much the
greater. This fact suggests the possibility that the total metabolism of the
declining population is higher than that of the growing population. If this is
true, the conditioning of the medium by the accumulation of metabolic wastes or
depletion of the dissolved oxygen would proceed at a greater rate in the water of
the old population than in that of the younger one, and the given numerical
density would exert a more severe effect upon the former than upon the latter.
This might account for the difference in the subsequent histories of the two
populations.
The question of comparative total metabolism was tested by determining the
relative rates of depletion of dissolved oxygen in the medium. Fifty adult
Daphnia chosen at random from a large culture were placed in each of six bottles
with 50 cc. of pond water containing Chlorella. A similar series was made up
using smaller animals from the same culture, 50 to the bottle. At the end of two
days the dissolved oxygen content of the water was determined by the Winkler
method, three bottles being required for each determination. The animals were
then transferred to fresh pond water, 50 cc. to the bottle, for a second two-day
period, after which the oxygen concentrations were again determined. The aver-
age depletion of dissolved oxygen per two-day period of conditioning was 1.19
p. p.m. in the water occupied by the 50 adults, 2.16 p. p.m. in the medium of the
50 young. In so far as the rate of oxygen depletion is a measure of metabolism,
the difference between these two figures indicates that the waning population of
larger biomass has a lesser, rather than a greater, total metabolism than the
waxing population of equal numerical size. Thus the continued decrease of the
old population cannot be assigned to a higher rate of conditioning of the medium.
There are two explanations for the continued decrease, the first of which is to
be found in changes in the age structure of the population. Since the individuals
of the declining population are of a greater average age than those of the in-
creasing population, their life expectancy is of course less. Thus the difference
in constitution of the waning population provides a reason for the higher daily
number of deaths in this phase of the cycle.
The second reason for the continued decrease in size of the population at
densities that formerly permitted increase is disclosed in the study of its previous
history as compared with that of the waxing population. The components of the
young, growing population had lived all of their lives, up to any density selected
for comparison, at population pressures lower than the given density. They had
never suffered severe crowding. But the individuals in the waning population of
TEMPERATURE AND DAPHNIA POPULATIONS 125
identical numerical strength have lived perhaps their entire lives at densities
greater than the present density.
A special experiment demonstrated that the life-long crowding experienced by
the latter individuals exerts a permanent adverse effect upon their reproductive
capacities. Animals were raised in a crowded condition until their first clutches
were laid in the brood chamber. They were then segregated, one animal to a
bottle, and their subsequent reproductive rates were compared with those of
animals reared in isolation. Unfortunately this early experiment was conducted
under conditions slightly different from those obtaining in the present population
studies: the volume of water used was 100 cc. rather than 50; and the temperature,
not controlled, varied between 22° and 27° C. The "crowded" state was a
population density of 25 animals/100 cc. Animals that lived under these con-
ditions as young (i.e. until the sixth day) and then were segregated, each into
100 cc., exhibited a reproductive rate only 62.7 per cent of that of animals that
had never suffered crowding.
It should be noted that this drastic effect was brought about by crowding of
only a very moderate intensity, as compared with that experienced by the declining
populations under discussion. Individuals whose previous history of crowding
has included population pressures ranging from 60 to 120 animals/50 cc. no doubt
suffer a far more severe inhibition of reproduction. Furthermore, it is believed
that subjection to high densities during early life has a lasting deleterious effect
upon survival as well as reproduction, which would help to explain the persistence
of a high death rate as well as a low birth rate in the shrinking population.
The effects of previous crowding may be sufficiently severe and persistent to
inhibit reproduction in the waning population completely and permanently. In
this event the population becomes extinct. As a rule, however, a few young are
produced toward the close of the cycle, pass their juvenile stages at minimal popu-
lation pressures, and attain maturity with their reproductive capacity unimpaired
by crowding. From these animals stems the next growth cycle of the population.
Oscillation consists essentially in the successive "overshooting" and "under-
shooting" of a theoretical equilibrium density. These phenomena appear to be
due to a delay, rather than a prolongation, in the manifestation of density effects.
The growing population withstands a high degree of crowding with a negligible
mortality. There is a lag before the effect of these population pressures is fully
felt upon the death rate, which, once raised, remains high for several days while
the decreasing population passes through formerly favorable densities. Likewise
the reproductive rate of the increasing population is at first unaffected by high
densities, but when finally checked, does not recover from the effects of crowding
until long after that state of crowding has ceased to exist. Thus overshooting is
occasioned by a delay in the expression of the adverse effects of high densities
upon reproduction and mortality, and undershooting results from a similar lag in
the manifestation of the beneficial effects of favorable densities.
C. Comparison of Oscillation at 25° and 18°.
The principal point of contrast in form of population curve at the two temper-
atures is the continued oscillation at 25° as compared with the tendency of the 18°
curves to approach an equilibrium value.
126
DAVID M. PRATT
Table I presents the duration, size-range, and mean size of the equilibria
established at 18° and at 25°, and the days of the respective population histories
that bounded these equilibria. After a single peak ranging from 184 to 241,
oscillation at 18° was greatly reduced in all cases, six of the populations achieving
nearly constant values (averaging 135.4) that they maintained until observations
ceased, whereas at the higher temperature equilibria were established on only
three occasions, and the general course of development was marked by a steady
increase, rather than a diminution, in both the amplitude and the period of
oscillation.
This progressive increase in the magnitude of oscillation at 25° is correlated
with a noteworthy decrease in the rate of population extinction. Of the 21
original populations, only seven survived until the third oscillation, but of these,
six were still flourishing when the experiment was discontinued. The reason for
TABLE I
Population equilibria at 18° and at 25° C.
Tempera-
ture
Population
Graph
Days bounding
equilibrium
Duration
Size
range
Mean
size
25°
Series A, No. 3
*
30-102
72 days
8- 30
21.9
Series A, No. 3
*
132-234
102 davs
30- 54
43.4
Series C, No. 5
I
50- 80
30 days
15- 28
22.5
18°
Series A, No. 1
*
110-174
64 days
124-145
133.6
Series A, No. 2
*
96-174
78 days
121-157
140.7
Series B, No. 5
*
112-172
60 days
127-156
140.4
Series C, No. 1
II
104-170
66 days
116-144
127.6
Series C, No. 2
II
100-170
70 days
129-157
143.3
Series C, No. 5
II
122-170
48 days
118-140
127.0
average of mean values for equilibria at 25° C. = 29.3
average of mean values for equilibria at 18° C. = 135.4
* Graph not presented in this paper.
the improved adjustment or heightened resistance to the environment apparent
in the latter half to two-thirds of population history at 25° is not clear.
Barring mutations, one cannot postulate genetic improvement through natural
selection, for all the animals were genotypically identical. Since the controlled
ecological conditions did not vary throughout the course of the experiment, one is
led to suspect some environmental factor that was not controlled. Of these,
there is only one which could conceivably have evoked the observed effect. As
previously stated, no attempt was made to control conditions of light. The day-
light, received from a north window, varied from season to season. It is not
improbable that the amount of dissolved oxygen in the medium was an important
factor in population growth. The period in which the populations appear to have
been better adjusted or more resistant to their environments, beginning at the
end of April, coincided with the season in which a longer daily duration of effective
light enabled the food-alga Chlorella to produce a greater amount of oxygen. This
added daily increment of oxygen may have been sufficient to account for the
TEMPERATURE AND DAPHNIA POPULATIONS 127
greater success of the 25° populations in the late spring and summer months.
These experiments are to be repeated, at least in part, under more rigidly con-
trolled light conditions.
If this is the correct interpretation of the increase in amplitude and period of
oscillation at 25°, evidently the 18° populations were started too late in the season
to experience any such improvement in environmental conditions.
A second point of comparison is found in the nearly complete and simultaneous
population "overturns" at 25°, and the more continuous overlapping of genera-
tions at 18°. At the higher temperature the first peak in numbers was due
entirely to the reproduction of the seed animals, which produced several broods.
Typically the first generation animals died during the first population decrease,
before the appearance of the third generation. The latter individuals were not
produced in numbers sufficient to prevent further decrease. None of the first or
second generation animals remained at the inception of the second major increase,
which was brought about by the production of the fourth generation. Thus there
was a minimum of overlapping of generations. Similar population overturns,
more or less complete depending upon the depth and duration of the depressions,
occurred between all the subsequent peaks.
In contrast, the course of development at 18° after the major upswing was not
thus punctuated by the simultaneous mass replacement of one generation by the
succeeding generation. A significant feature of population history at the lower
temperature was the accumulation of successive generations. The simultaneous
presence of animals of all ages insured a steady replacement of adults and resulted
in a sustained continuity in growth and maintenance never observed at 25°.
The lack of a sufficient number of steadily reproducing adults in an 18° popu-
lation occasions the spasmodic type of population growth witnessed in the first
30 or 40 days of development. This was the period during which the second
generation was being produced. The relatively infrequent production of young
by the two seed animals and a comparatively high infant mortality result in a
highly irregular curve. The attaining of maturity by animals of the second
generation caused the tremendous increase in population size which began on
about the 40th day. From this point on, the overlapping of successive genera-
tions and the constant replacement of producers gives the curve its characteristic
unbroken continuity.
It should be recalled that oscillation at 25° results from an alternation of
fluctuations, approximately equal in amplitude, in the number of births and the
number of deaths per day. The mechanism of oscillation at 18°, in terms of the
births/deaths ratio, can be analyzed in Graph IV, which is similar in purpose and
in method of construction to Graph III. The data are those of population No. 1
in Graph II.
Examination of these curves reveals that there was far greater variation in the
number of births per day than in the number of deaths. Moreover, the two major
changes in the size of the population, viz. the tenfold increase between the 40th
and 56th days, and the later more gradual decrease, were correlated, respectively,
with the periods of maximal and minimal numbers of daily births. While it must
be conceded that the number of deaths per day was slightly greater while the
population decreased than during the period of increase, both of these levels on
the deaths curve are equalled in other parts of that curve, and the difference be-
128
DAVID M. PRATT
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TEMPERATURE AND DAPHNIA POPULATIONS 129
tween them is immaterial when compared with the variations observed in the
births curve. Thus the principal changes in the size of the population are
attributable to fluctuations in the number of births, while the number of deaths
per day remains approximately constant. This mechanism of oscillation should
be contrasted with the alternating fluctuations, approximately equal in amplitude,
in the numbers of daily births and deaths that constitute the mechanism of
oscillation at 25°.
The lowering of the temperature to 18° exerts a damping effect upon the magni-
tude and persistence of oscillation that characterizes population development at
25°. At either temperature the waxing population reaches a' size that it is in-
capable of sustaining indefinitely. Whereas at 25° this maximum was generally
less than 100 and never exceeded 126, all the 18° populations attained peaks ap-
proximately twice as high, covering the range 184-241. At 25° the effects of
previous crowding upon both reproduction and longevity manifest themselves
quite suddenly, and a sharp peak is described. At the lower temperature
crowding acts upon reproduction alone, and its full expression is delayed longer
than at 25°, with the result that the population maintains its maximum long
enough to describe a short "plateau." Furthermore, whereas the 25° curve sinks
almost to the baseline before increase is again possible, the waning 18° population
succeeds in halting its decrease at a density which it can maintain, with minor
oscillations, for at least 78 days. It should be noted that this density is greater
than that achieved in any of the 25° maxima. Thus regardless of temperature,
the waxing population overshoots the density of potential stabilization, but the
waning population at 18° does not undershoot it as the 25° population does. This
virtually terminates oscillation at the lower temperature after the first peak, in
sharp contrast to the continued and progressively increased oscillation at 25°.
INFLUENCE OF TEMPERATURE UPON LONGEVITY AND REPRODUCTION
To account for the observed differences in the histories of populations at 18°
and 25°, a series of experiments was undertaken to determine the effect of these
temperatures upon the two primary variables in population growth, namely dura-
tion of life and the reproductive rate. Since such an investigation must take into
consideration the influence of population density if it is to accomplish its ultimate
purpose, the experiments were so designed as to measure, at each of the tempera-
tures tested, the mean longevity and reproductive rate of animals living at different
constant densities.
The desired number of parthenogenetically produced female Daphnia were
placed in 50 cc. of fresh pond water with Chlorella added, not more than 12 hours
after their release from the maternal brood pouch. The medium was renewed at
two-day intervals, and the dead animals and young, when they appeared, were
removed and counted daily. Population pressures of more than one animal per
bottle were maintained constant by the introduction of substitute animals to take
the place of those that had died. The problem of distinguishing these "substi-
tutes" from the extant original members of the controlled population was sur-
mounted by staining them with Neutral Red, a vital stain which in concentrations
sufficient to dye the animals apparently did not injure them. (When fed only
Chlorella, Daphnia magna does not develop the rich red color generally charac-
130
DAVID M. PRATT
teristic of the species.) A staining period of 12 to 24 hours in pond water tinged
with a few drops of a concentrated Neutral Red solution rendered the animals
distinguishable from untreated individuals for several days. Two precautions
were exercised in the selection of substitutes: 1. they were matched for size with
the original members still living in the population, and 2. only individuals without
eggs were chosen for this purpose. Thus, in so far as it was possible to estimate
it, the substitutes' contribution to the total density effect was proportional to
their number, and all of the young produced in the population were born of
charter members.
A. Experiments at 25°.
Thirty tests were made at a density of one animal per 50 cc., four at densities
5 and 10, and two at densities 25, 50 and 75.
The survival curve of the 30 single animals and the average survival curves
for the five higher densities are plotted on Graph V. To facilitate a quantitative
' *v
3O 40
TIME IN DAYS
GRAPH V. Survival at different constant population densities, 25° C.
Legend: population density 1
population density 5 -o-o-o-o-o
population density 10
population density 25 -• •-• — •-
population density 50 -
population density 75 -
comparison of survival at the six densities tested, the total number of animal days
lived by each population was divided by the number of original members to give
the mean duration of life at each population pressure. These data are plotted
on Graph VI, which discloses the noteworthy fact that the greatest mean longevity
occurs in populations of five, rather than at the minimal density, and that animals
even at a density of ten per bottle lived longer, on the average, than did those in
isolation.
Two interpretations of this phenomenon suggest themselves. MacArthur and
Baillie (1929) have developed the thesis that the mean longevity of Daphnia magna
is an inverse function of the metabolic rate and have reported (1929a) that
TEMPERATURE AND DAPHNIA POPULATIONS
131
metabolic rate as indicated by the rate of heart beat is inversely proportional to
population density in the range 1 to 25 animals per 100 cc. These authors did
not determine the influence of population density upon longevity in Daphnia, but
they implied that the reduced metabolic rate evinced by crowding might exert the
same effect upon longevity as a metabolism lowered by some other process, such
as decreasing the temperature. According to this theory, then, increased popu-
lation pressure, up to the point of actual injury, might be expected to prolong
life. MacArthur and Baillie's hypothesis may give the correct interpretation of
the occurrence, observed in the present experiments, of the maximal longevity at
a supraminimal density.
There is, however, a second possible explanation for this phenomenon. It was
noted that the water in bottles containing only one animal was usually slightly
clouded with bacteria, whereas the medium of larger populations was always kept
30 60 90 1
POPULATION DENSITY
GRAPH VI. Population density and mean longevity.
Legend: 18° C. -
25° C. -
clear by the feeding animals. The bodies of the isolated individuals, when found
dead, were frequently covered with a bacterial slime, which was occasionally ob-
served even before death, in severe cases greatly hindering the animal's move-
ments or even imprisoning it completely. The slime was composed of motile rods
and spirilla — common fresh water saprophytes — and their gelatinous secretion.
Since its appearance upon a live animal in macroscopic proportions almost in-
variably signalled the death of the animal within a day or two, it is believed to
have contributed to the relatively high death rate at the minimal density. More
crowded populations apparently never suffered from this effect; their greater
numbers enabled them to maintain control of the bacterial flora.
This explanation is reminiscent of one proposed to account for a similar relation
between density and mean longevity observed in populations of a different animal.
Alice (1931) has suggested that the positive correlation of mean longevity with
population pressure in Drosophila in the density range of 1 to 35 or 55 flies per one
132
DAVID M. PRATT
ounce bottle, reported by Pearl, Miner and Parker (1927) may be due to the
inability of the smaller populations to keep in check the "wild" yeasts con-
taminating the cultures.
The available evidence does not warrant a decision between the two interpre-
tations, metabolic and bacterial, of the results recorded here. It is not improbable
that they are both operative in the present case.
The reproductive rate of each population was calculated by dividing the total
number of young produced by the number of animal days lived. Graph VII, in
which the results are presented, shows that reproductive rate is an inverse func-
tion of density throughout the range in which it was determined, and it drops
most rapidly as the density is increased to 25 animals per bottle.
These experiments, yielding quantitative measurements of the effects of vari-
ous constant densities upon longevity and reproductive rate, offered an oppor-
tunity for studying the nature of the density effect as the limiting factor in
a eo so
POPULATION DENSITY
GRAPH VII. Population density and reproductive rate.
Legend: 18° C. -
25° C.
population growth. In population studies with different animals a variety of
density effects have been described, but almost certainly the only influence
crowding can exert upon either the birth rate or the death rate of a parthenogenetic
form such as Daphnia is through the conditioning of the medium. This might
lead one to expect that medium which had been heavily conditioned would exert
the same adverse effects upon reproduction and longevity as those brought about
by actual crowding. To test this, individual Daphnia were reared in isolation in
the water conditioned by the populations of different constant densities. Every
other day their medium was renewed with that which one of the larger populations
had been conditioning for the past two days. Contrary to expectation the
isolated animals suffered thereby no impairment of reproductive capacity. Thus
the conditioning of the medium is only temporary, and probably consists in the
accumulation of some volatile inhibitory substance, such as carbon dioxide, or the
depletion of the dissolved oxygen supply.
Further experiments have been planned to ascertain more exactly the nature
TEMPERATURE AND DAPHNIA POPULATIONS
133
of this limiting factor. Moreover, individual Daphnia are to be raised in medium
effectively connected with that of animals living under crowded conditions, so as
to test the possibility of a density effect induced directly by crowding per se,
rather than indirectly through the conditioning of the medium.
B. Experiments at 18°.
Twenty-five tests were run at a density of one animal per 50 cc., two at
densities 5 and 10, and one at density 135. On the 81st day of observation, when
these experiments had to be discontinued for lack of time, all the animals at the
minimal density were dead, but some individuals were still living at each of the
three higher densities. The data are therefore complete for density 1 but must be
regarded as partial only for densities 25, 75, and 135. Had the experiments gone
to completion, the mean longevities for the latter three densities obviously would
GRAPH VIII. Survival at different constant population densities, 18° C.
Legend: population density 1
population density 25
population density 75
population density 135
have been higher than those obtained, and it is impossible to say whether the
average reproductive rates would have differed appreciably from those observed,
and in which direction. Of the two sets of data, those concerning reproduction
may perhaps be considered the more accurate.
The range of densities tested was extended to include the mean equilibrium
value, 135 (see Table I, p. 126), in the 18° populations. The survival curve of the
25 single animals and the average survival curves for the other densities are
plotted on Graph VIII.
Calculation of the mean longevity by dividing the total number of animal
days by the density of the population yields results which, for densities 25, 75 and
135 are obviously only minimal, since some animals were still living at those
densities when observations ceased. The data are shown in Graph VI.
The mean reproductive rates for the 81 days of observation are presented in
Graph VII.
134 DAVID M. PRATT
C. Comparison of results, 25° and 18°.
Throughout the range of population densities tested, duration of life is greater
at 18° than at 25° (Graph VI). This results from the positive correlation of
metabolic rate with temperature and the negative correlation of longevity with
metabolic rate.
The action of population pressure is quite different at the two temperatures.
Whereas a density of 5 was found to be the optimum for duration of life at 25°,
throughout the density range 1 to 75 longevity at 18° is a direct function of
population pressure, and animals living at density 135 lived, on the average, longer
than did those in isolation. The bacterial contamination of the medium which
is believed to have contributed to the death rate of single individuals at 25° was
never observed in the 18° bottles. This is, then, an indirect effect of temperature
upon duration of life: even at the minimal density the colder water did not support
a bacterial flora sufficiently concentrated to injure the animals. The absence of
a bacterial effect at 18° makes it seem likely that the positive correlation of
density and longevity in this case is incidental to a lowered metabolic rate. At
18° the population pressure at which metabolism is depressed to the point of
positive injury lies somewhere in the density range 25 to 135, perhaps at about 75
animals per bottle.
Whereas a decrease in temperature of 7° raised the mean longevity, presumably
through depression of the metabolic rate, it did not thereby bring about an equiva-
lent reduction in reproductive rate. Apparently the rates of reproduction and
mortality are not dependent upon exactly the same physiological processes. If
they were, a given increase in longevity with a reduction in temperature would be
correlated with a decrease in birth rate of the same magnitude.
The action of increasing density upon reproductive rate is very similar at the
two temperatures (Graph VII), although it is slightly more severe at 25°: at the
minimal density birth rate at 25° is higher than at 18°, but drops faster with
increased crowding and at density 25 is slightly lower than the corresponding
18° rate.
The relative potential rates of population increase (i.e. the rates that would
obtain if there were no density effects) can be calculated by comparing the data
for the minimal density at the two temperatures. The birth rate at 18° is 2.19
young per animal day, or a gross factor of daily increase of 2.19X. The death
rate (which is the reciprocal of the mean longevity, or 1/47.6) is .02 IX per day.
Thus the net rate of potential daily increase (2.19X-.021X) is 2.17X. At 25°,
gross increase (2.38X) minus death rate (.044X) yields a net rate of potential daily
increase of 2.34X. When one considers that birth rate at 18° is only slightly less
than at 25° and that longevity at 18° is more than twice as great as at 25°, this
result is perhaps astonishing, but it illustrates the fact that birth rate is so much
greater than death rate as to be the only effective factor in the net rate of increase.
The reproductive rate taken alone gives the 25° population an initial advantage
of .19X (2.38X-2.19X) over the 18° population. Granting the observed 25°
death rate of .044X, the net rate of potential increase at 18° could not equal that
at 25° even if death rate at the lower temperature were reduced to zero. But, as
we have seen, the differential action of population density is such that ultimately
a population attains a greater size at 18° than at 25°.
TEMPERATURE AND DAPHNIA POPULATIONS 135
It will be recalled that oscillation at 25° was brought about by an alternation
of approximately equivalent effects of population density upon the number of
births and the number of deaths. The results of the reproduction and longevity
experiments at 25° bear out the contention that population density at that temper-
ature affects both of the primary variables in population growth, in opposite
directions and to approximately the same extent. On the other hand, the
reproduction and longevity experiments at 18° give results consistent with the
observation that the mechanism of oscillation at that temperature was the
fluctuation in the number of births about a nearly constant daily number of
deaths. Population pressure has a relatively insignificant effect upon mortality.
Moreover, if the number of deaths per day in an increasing population remains
constant, the death rate must be an inverse function of population density. This
deduction is supported by the fact that mortality in the reproduction and longevity
experiments was lower at density 135 than at the minimal population pressure.
This action of density upon death rate, operating in generally the same direction
as the effect of density upon birth rate, tends to moderate, rather than intensify,
the severity of oscillation. Therefore oscillation at 18° must be attributed
wholly to changes in birth rate.
From the reproduction-longevity data one can calculate theoretical asymptotes
for populations at 18° and at 25°. The number of young produced by an indi-
vidual of mean longevity and reproductive rate at a given density can be de-
termined by dividing the total number of young born at that density by the size
of the population. These figures for the four population pressures tested at
18° C. are as follows:
density 1 25 75 135
average number of young individual 104.5 10.7 0.73 0.19
Obviously a population of such density that each member could just replace itself
before dying should be capable of maintaining a constant size. It is found by
interpolation that the density at which the average animal produces one young
in the course of its life is 73.6. It should be noted, however, that this theoretical
asymptotic value is considerably lower than the mean of equilibrium values
(135.4) actually established in the 18° populations. The discrepancy is serious,
and perhaps cannot be entirely explained by the fact that the reproduction-
longevity experiments did not go to completion.
At the higher temperature the agreement between observed equilibrium values
and the theoretical asymptote is much closer. The number of young produced per
individual in the 25° reproduction-longevity experiments is as follows:
density 1 10 25 50 75
average number of young individual 53.9 26.7 23.1 0.82 0.26 0.08
The calculated asymptote is 24.8 animals per bottle, while the mean of equilib-
rium values actually observed in the 25° populations is 29.3.
The explanation for the discrepancies between observed and calculated equi-
librium values is not clear. It is suggested that the age-structure of the popula-
tion is a significant factor. Apparently the conditions implied by a density of a
given number of animals of the same age are different from those implied by a
density of the same number of animals of different ages. Although the repro-
136 DAVID M. PRATT
duction-longevity experiments at both temperatures yield theoretical equilibrium
values that are probably lower than the actual levels of stabilization, these ex-
periments undoubtedly give a faithful picture of the relative effects of different
densities upon the reproductive rate and upon longevity, and the data for the
minimal densities can be regarded as absolute, under the given conditions.
DISCUSSION
A. Oscillation.
Fluctuations in the density of populations in nature can usually be assigned
to changes in environmental forces, which may be physico-chemical or biotic.
The environmental disturbance may evoke an immediate response in the numbers
of the species under consideration (as in the case of epidemics, sudden changes in
meteorological conditions, etc.), or its action may be delayed for a longer interval.
An example of this second category is the determination of the future size of adult
populations of marine fishes by the effects of various environmental agencies upon
the early developmental stages (Hjort, 1914; Johnstone, 1928). Presumably the
periodic oscillations in the numbers of fur-bearing mammals and game birds which
have been synchronized with sunspot cycles (Elton, 1924; Gross, 1931; Naumov,
1939; Braestrup, 1940; Green and Evans, 1940) result from complexes of environ-
mental vectors whose action is more or less delayed.
When one considers the instability of the environment, it is not surprising that
natural populations undergo violent fluctuations. However, it has been argued
from mathematical grounds that the interaction of two or more animal species,
e.g. predator and prey (Volterra, 1926) or parasite and host (Nicholson, 1933) is
such as to give rise to rhythmic pulsations in the numbers of the animals, even
though the environment is maintained constant in all other respects. In such a
situation, oscillation in population density would be attributable to biotic forces
exclusively, the physico-chemical factors of the environment being fixed.
Of the cases of fluctuations in numbers whose cause has been ascertained, all
that have come to my attention are laid to variations in some external agency.
The oscillations in Daphnia populations discussed in this paper are of an essen-
tially different nature. Here the agent of fluctuation is internal and intrinsic.
While the environment plays an important role, it is an environment whose
critical changes are determined by the activities of the animals themselves. The
cause of oscillation is the delay in the action of population density upon mortality
and the reproductive rate, rather than a variation in some external environmental
agency. It is obvious that fluctuation would not occur if the effects of a given
density upon birth and death rates manifested themselves immediately; an in-
creasing population would gradually develop an asymptote instead of "over-
shooting." Thus the ultimate source of oscillation is a lack of synchronization of
a physiological state with the forces that provoke it.
B. Influence of temperature on population size.
It was originally intended to obtain a quantitative expression of the influence
of temperature on population size by comparing the asymptotes developed at the
different temperatures. Since this is clearly impossible, apparently the most
satisfactory comparison would be one involving the mean sizes of the populations.
TEMPERATURE AND DAPHNIA POPULATIONS 137
The average sizes of the 21 populations at 25° covered a range of 18.5 to 43.0, with
the average at 32.6. The range of average sizes of the 16 populations at 18° was
104.7 to 126.2, the mean 112.4. Thus the mean of population size at 18° was
about two and one half times as great as at 25°.
This result is consistent with the common experience that populations, and
particularly those of marine plankton, attain greater densities in cold than in
warmer regions (Oltmanns, 1923; Belehradek, 1935; Welch, 1935; Russell and
Younge, 1936; Hesse, Allee and Schmidt, 1937). It should be borne in mind that
this greater abundance in polar waters refers to the size of the equilibrium popula-
tion, rather than to the productivity in terms of the rate of turnover.
Inasmuch as the cause of this relative abundance is still not certain, and a
variety of theories have been developed to account for it, the possibility of ap-
plying the results of the present study to the problem should be of considerable
interest. At the outset, however, it is apparent that the type of environmental
factor preventing unlimited growth in these experimental populations (i.e. a
biological conditioning of the medium by the accumulation of metabolites and/or
depletion of the oxygen supply) is probably never an effective limiting factor in
the open ocean. In recent years, however, it has been contended that the
latitudinal variation in plankton abundance so often observed in the sea obtains in
fresh waters as well (Welch, 1935), and it is quite possible that the limiting factor
in the Daphnia populations is operative in some fresh water situations.
Of the various hypotheses advanced by the oceanographers, there is but one
which might be applicable to the present case. This is the theory that attempts
to explain the greater asymptotic level of polar planktonic populations by a direct
effect of temperature upon the metabolic rate. It is argued that the lower
metabolism in cold waters results in a longer duration of life and thus in an
accumulation of generations; and further, that this increase in longevity more than
offsets the concomitant reduction in reproductive rate. In short, the decrease in
temperature exerts a greater effect on duration of life than upon the birth rate.
Loeb (1912) supported this argument with the observation that the prolongation
of life of sea urchin eggs with a drop in temperature greatly exceeded the retarda-
tion of their development. The theory involves only the direct effects of temper-
ature upon birth and death rates. It alleges to explain the observed results
without reference to any action of population density upon reproduction and
duration of life.
A critical examination of the Loebian theory discloses that it really cannot
account for differences in asymptotic levels. The disproportionately greater
longevity at the lower temperature cannot possibly influence the height of the
asymptote, since birth and death rates in a population that has attained an
asymptote are equal. The equilibrium level is determined by two factors: the
previous rate of increase of the population and the duration of that increase. The
birth rate/death rate ratio determines the rate of population increase. It is in
this ratio that the disproportionately great longevity at the lower temperature
would express itself, yielding a greater net rate of population increase in colder
than in warmer waters. But the second factor, namely the duration of population
increase, is in no way affected by the birth rate/death rate ratio. It is determined
by some limiting factor in population growth other than temperature. This
limiting factor may, in turn, be influenced by temperature, but it is essentially a
138 DAVID M. PRATT
result of population density. Without it, that is with no limit to the duration
of increase, the population would continue growing, geometrically and indefinitely.
Since Loeb's theory involves only the rate of increase and disregards the factor of
duration of increase it makes no provision for any check in population growth.
The inescapable conclusion is an everlasting logarithmic increase. From a
slightly different point of attack this criticism may be rephrased thus. Since
there are two factors involved in the asymptotic level ultimately attained, a
population with an infinitesimally low rate of increase may eventually reach a
greater asymptote than that developed in shorter time by another population with
a much higher rate of increase. As an example, in the present experiments the
potential rate of increase at 25° was found to exceed that at 18°, yet populations
at the latter temperature attained the greater mean size. It should be pointed
out, however, that owing to seasonal phenomena, the time element may be
critical in the development of some populations in nature. Because of the
brevity of the favorable season, these populations may never reach their potential
asymptotes. In this case the rate of increase is the all-important factor in the
size of the population at any given moment.
The explanation for the greater mean size of the 18° populations would appear
to be a differential action of density at the two temperatures. The reproduction-
longevity experiments revealed that increasing population pressure exerts a more
severe effect upon the birth and death rates at 25° than at 18°. Possibly this
result is related to the difference in solubility of atmospheric oxygen in the medium
at different temperatures, but whatever the nature of the conditioning may be, the
influence of temperature upon mean population size is indirect. It operates
through the differential effects of population pressure. Thus the difference in
temperature exerts its observed influence upon the mean of population size only
by modifying the action of population density.
SUMMARY
1. The development of populations of Daphnia magna was followed at two
different constant temperatures. Sixteen populations were maintained at 18° and
21 at 25° C. The 50 cc. of pond water which served as medium were renewed
every other day and always contained an excess quantity of the food-alga Chlorella.
2. Population development at 25° proved oscillatory in nature, four peaks
occurring in 234 days, with a maximum population size of 126 animals. In the
174 days of observation at 18°, one major peak was observed (maximum 241)
followed by a decrease and virtual stabilization at a population density of
about 135.
3. Analysis of the oscillation disclosed that it is due to a delay in the expression
of the effects of population density upon birth and death rates.
4. The mechanism of oscillation at 25° is an alternation of fluctuations in
numbers of births and numbers of deaths. The mechanism at 18° is the fluctua-
tion in the number of births about a nearly constant number of deaths.
5. Experiments with a series of population densities artificially maintained
constant showed that birth rate at 25° is an inverse function of population density.
At 18° the effect of density is similar but less severe.
6. Under these conditions of constant density, mortality at 25° is in general a
function of population density, although the minimal mortality occurs at a
TEMPERATURE AND DAPHNIA POPULATIONS 139
density of 5. At 18° mortality is but little affected by conditions of density, and
is apparently least at about 75 animals/50 cc.
7. The mean of population size at 18° was two and one half times as great as
that at 25°.
8. This fact is compared to the supposed greater density of planktonic popula-
tions in polar than in tropical waters. The results of this study cannot be applied
to the problem of marine plankton abundance since the limiting factor in the
present case (the conditioning of the medium by the accumulation of metabolites
and/or depletion of the dissolved oxygen supply) is presumably never operative in
the ocean, although it may be operative in some fresh water situations.
9. The possibility of accounting for the greater mean size of the 18° populations
by reference to the direct effect of temperature upon longevity is considered but
rejected. A basic fallacy is pointed out in the theory which attempts to explain
by such a direct effect of temperature the greater density of asymptotic popula-
tions in polar than in tropical regions.
10. It is concluded that the influence of temperature upon mean population
size observed in these experiments is indirect: the temperature difference exerts
its effect only by modifying the action of population density.
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RATE OF BREAKING AND SIZE OF THE "HALVES" OF THE
ARBACIA PUNCTULATA EGG WHEN CENTRIFUGED IN
HYPO- AND HYPERTONIC SEA WATER
ETHEL BROWNE HARVEY
(From the Marine Biological Laboratory, Woods Hole, and the
Biological Laboratory, Princeton University}
PROBLEM
Arbacia punctidata eggs, when centrifuged in a mixture of sea water and
isosmotic cane sugar solution used in the proper proportion to keep the eggs sus-
pended during centrifugation, break quite uniformly into "halves" of definite size
(E. N. Harvey, 1931; E. B. Harvey, 1932-1941). The question arises as to
whether the eggs break more or less readily in hypotonic solutions than in sea
water, and whether the relative size of the two "halves" remains the same, that
is, whether the extra water is distributed equally in the two halves. It has been
shown by Lucke (1932b, 1940) that when the eggs are broken into halves in sea
water first and the halves are then placed in hypotonic sea water, both halves
swell but the heavy (red) half swells a little less than the light (white) half owing
to the presence of more of the osmotically inactive material (yolk granules) in this
(red) half. The present problem is concerned with centrifuging the eggs after
they have been swollen in hypotonic sea water. It has been shown (E. B. Harvey,
1941 ) that the rate of breaking and the relative size of the two halves varies with
the amount of centrifugal force used. With a force of 10,000 X g, which I have
taken as a standard force throughout my experiments, the white (centripetal)
half is slightly larger than the red (centrifugal) half. With a greater force, the
red half is larger while the white half is correspondingly smaller. With a smaller
force, the red half is smaller than with greater forces, and the white half corre-
spondingly larger. In the present experiments, therefore, a uniform standard
force was used, 10,000 X g.
The size of the halves obtained by centrifuging the eggs in hypo- and hypertonic
sea water and subsequently returning them to normal sea water was also studied,
in order to determine how the normal water balance was regained.
METHODS
Before centrifuging, the eggs from one female were kept for a half hour in 60
per cent, 80 per cent, 100 per cent (control) and 125 per cent sea water, a sufficient
time for them to attain equilibrium with the medium. Eggs kept for six hours in
the solutions showed no appreciable further change in size. The sugar solutions
added to the sea water to keep the eggs suspended during centrifugation, were
made up of the same tonicity as the hypo- and hypertonic sea water. The four
tubes containing 60 per cent, 80 per cent, 100 per cent (control) and 125 per cent
sugar-sea water solutions were all centrifuged at the same time in each experiment;
141
142 ETHEL BROWNE HARVEY
each tube contained one part of the egg suspension to three parts of the corre-
sponding sugar solution, this being the proper proportion to keep the eggs sus-
pended and free to break during centrifugation. The unbroken eggs in all the
tubes come to lie at the same level, so that they are all subjected to the same
centrifugal force which, of course, varies with the radial distance of the layer
of eggs from the axis of the centrifuge, according to the equation F — .04
X R (= radius in cm.) X (R.P.S.)2. It is necessary to use the eggs from only
one animal for one experiment since there is considerable variability in size,
segregation of granules and ease of breaking in eggs from different females, but
those from one female are remarkably constant in this respect. The eggs were
centrifuged for three to six minutes at 10,000 X g, according to the ease of
breaking of the particular batch of eggs, and were then placed in dishes of sea
water of the corresponding tonicity. The measurements were made with an
ocular micrometer and checked in several experiments with a filar micrometer; the
figures are accurate to about 0.6 IJL. The measurements recorded are the average
of ten cells, made with an optical equipment giving a magnification of 400 times;
the eggs lay free in the media in Syracuse watch glasses.
The experiments were performed many times with the same general results.
The data obtained in a typical experiment are given in Table I A, B, C. The
same eggs were used throughout the experiment.
RESULTS
Rate of breaking (Table I A)
The rate of breaking into halves may be judged by the number of broken eggs
in comparison with the number of whole, unbroken eggs obtained after centrifuging
for a definite time with a definite force. When tubes containing suspensions of
eggs in a 60 per cent sea water-sugar medium, 80 per cent, 100 per cent (control)
and 125 per cent were centrifuged at the same time, usually for four minutes at
10,000 X g, the degree of breaking increased in the order named. In most of the
experiments, practically all the eggs were broken in the 125 per cent medium
while very few were broken in the 60 per cent medium. An average experiment
(Table I A), gave 10 per cent of the eggs broken in the 60 per cent medium, 20
per cent in the 80 per cent medium, 70 per cent in normal sea water and practically
all in the 125 per cent medium. In an experiment where only 50 per cent of the
eggs were broken in the 125 per cent medium, none were broken in the 60 per cent
medium. The eggs break, therefore, less readily in hypotonic sea water, and
more readily in hypertonic sea water, than they do in normal sea water.
Size of the halves (Table I A; Photographs, Plate I)
When eggs are swollen in hypotonic sea water or shrunken in hypertonic sea
water and then centrifuged, the increase and decrease in size is almost entirely in
the white halves, the red halves being nearly the same size as those centrifuged in
PLATE I
Photographs of living Arbacia punctulata eggs centrifuged in hypo- and hypertonic sea water,
and the controls in normal sea water, and the halves into which they break with a force of 10,000
X g for four minutes. Magnification approximately 275 X, all magnified exactly the same.
EGGS CENTRIFUGED IN ANISOTONIC SEA WATER
143
60^
/°
'
10
8
Control 100$
12
PLATE I
144
ETHEL BROWNE HARVEY
normal sea water (Table I A). When the eggs are centrifuged in 60 per cent sea
water, the white halves are very much larger than those obtained in the control
(100 per cent sea water), whereas the red halves are only slightly larger than in the
control (Cf. Photographs 2, 3 with 8, 9). When centrifuged in 80 per cent sea
water, the white halves are somewhat larger than in the control, the red halves
almost the same size (Photographs 5, 6). When centrifuged in hypertonic sea
TABLE I
Sea
water
Whole egg
Diani. /* Vol. ^3
White half
Diam./j Vol.^3
Red half
Diam.M Vol./i3
Nucleus
Diam. n Vol. M3
Per
cent
broken
A. Eggs in hypo- and hypertonic sea water, then centrifuged
60%
82.4 (292,900)
70.4 (182,700)
58.0 (102,200)
16.0 (2,145)
10%
80%
74.9 (220,000)
62.1 (125,400)
56.3 ( 93,400)
12.8 (1,098)
20%
100%
72.0 (195,400)
59.0 (107,500)
56.0 ( 91,950)
11.5 (796)
70%
125%
66.6 (154,700)
51.7 ( 72,360)
53.8 ( 81,540)
9.6 (382)
98%
B. Recovery in 100 per cent sea water
60%-100%
80%-100',
100%
125%-100%
72.0 i (195,400)
72.0 1 (195,400)
72.0 (195,400)
72.01 (195,400)
59.2 (108,600)
59.2 (108,600)
59.0 (107,500)
56.3 ( 93,940)
55.7 ( 90,480)
54.7 ( 86,170)
56.0 ( 91,950)
57.6 (100,060)
11.2 (736)
11.5 (796)
11.5 (796)
11.2 (736)
C. Eggs centrifuged in sea water, then placed in hypo-hypertonic sea water
100%- 60%
82.4
(292,
900)
67.4
(160
,300)
63
A
(133
,400)
100%- 80%
74.9
(220,
000)
61.8
(123
,600)
57
.2,
( 97
,990)
100%
72.0
(195,
400)
59.0
(107
,500)
56
0
( 91
,950)
100%-12S%
66.6
(154,
700)
54.4
( 84
,300)
51
2
( 70
,300)
D. Lucke' s (I932b) 2 mean values for C (above)
100%- 60%
(84.6)
317
,380
(69.5)
175
,560
(63
-9)
136
,700
100%- 70%
(80.6)
274
,020
(66.3)
152
,320
(61
.7)
123
,060
100%
(72.2)
197
,440
(59.2)
108
.600
(55
.7)
90
,680
1 These are not actual measurements because of lack of time to measure these in the same
experiment as the rest. The return to normal size is approximately perfect, as determined in
other experiments and as found also bv Lucke and co-workers, who publish their measurements
(1931a, p. 402).
2 Only the volumes are given by Lucke; the diameters are calculated from the volumes.
water (125 per cent) the white halves are much smaller than in the control, the red
halves about the same size as the controls, sometimes larger, sometimes a little
smaller; the white halves are now in most experiments smaller than the red halves
(Photograph 12); in a fewr experiments they were the same size. In the controls,
the white halves were always considerably larger than the red halves (Photo-
graphs 8, 9).
Unusual batches of eggs occur occasionally, as noted in previous papers
EGGS CENTRIFUGED IN ANISOTONIC SEA WATER 145
(1936, 1941), in which, when centrifuged in normal sea water with the standard
force, the red half is very small, and the white half correspondingly very large, in
the ratio of 8 : 1 by volume. When such batches of eggs are centrifuged in 80
per cent sea water, the halves are of approximately the same relative size as in
normal batches, in the ratio of 4 : 3 by volume, as noted previously (1941).
When the unusual batches are centrifuged in hypertonic sea water, on the other
hand, the relative inequality in the two halves remains; that is, the red halves
are very small.
Stratification of whole eggs, and content of halves (Photographs, Plate I)
As would be expected, the granules pack much more when the eggs are
centrifuged in hypotonic media than in normal sea water (Photographs 1, 4, 7).
The packing of the granules takes place to such an extent in the 60 per cent sea
water that the clear layer is very large and usually the white halves (Photo-
graph 2) are almost entirely free of granules, all of them having been thrown down
into the red half, although, as stated above, this red half is not much larger than
the red half obtained in normal sea water which contains none of the mitochondria
and only part of the yolk. This can be beautifully demonstrated in eggs stained
with the vital dye, methyl green, which selectively stains the mitochondria. The
purple-staining mitochondria are all in the red half. In the whole egg centrifuged
in 60 per cent sea water, the mitochondrial layer is very thin, being spread over a
greater area. The pigment granules are so well packed in the hypotonic solutions
that the line of demarkation between yolk and pigment is very sharp, much more
so than in eggs centrifuged in normal sea water. When the eggs are centrifuged
in 60 per cent sea water, many of the white halves and also the upper portion of
the whole eggs containing the clear layer burst soon after centrifugation; the red
halves and red portion of the whole egg remain intact. This bursting is probably
due to the thinness of membrane which presumably decreases in thickness as the
area it covers increases.
When centrifuged in hypertonic sea water, the clear layer is small, the
mitochondrial layer very thick, being spread over a small area, and in many cases
is very well marked (Photograph 11). The white half is thus quite granular.
The pigment is not well separated from the yolk, there being no clear line of
demarkation. It is obvious from Photograph 11, that it is in general not accu-
rate to speak of "well-stratified" eggs, since they may be well-stratified with re-
spect to the mitochondria and poorly stratified with respect to the pigment and
yolk. Many batches of eggs occur, in which, when centrifuged in normal sea
water, the mitochondrial layer is indistinguishable while the yolk and pigment
layers are well formed. In typical batches of eggs, however, the stratification in
normal sea water is intermediate between that obtained in hypotonic sea water
and in hypertonic sea water (Photograph 7).
Recovery in normal sea water (Table I B; Photographs, Plate II)
When whole normal eggs are swollen in hypotonic sea water or shrunken in
hypertonic sea water, and are then returned to normal sea water, they return to
normal size, as shown previously (for hypotonic) by Lucke and co-workers
(1931a; 1932a). The same holds for centrifuged whole eggs. The two half-eggs
146 ETHEL BROWNE HARVEY
obtained by centrifuging an egg swollen in hypotonic sea water, shrink when
returned to normal sea water, but not at all to the same extent. The white half
loses a great deal of water, the red half very little (Photographs 13, 14). The two
halves become of approximately the same size as the two halves obtained from a
normal egg centrifuged in normal sea water (Photographs 8, 9). The loss of
water from the white halves takes place exceedingly rapidly. Lucke and co-
workers (1927; 1931b; 1932a) have called attention to the much more rapid
shrinking than swelling in the case of whole eggs.
When whole normal eggs are shrunken in hypertonic sea water, and returned
to normal sea water, they likewise regain their normal size. When eggs are
centrifuged in a hypertonic solution (125 per cent), the two halves, as stated
above, are of nearly equal size, the red half being in most batches a little larger
than the white half (Photograph 12). When these halves are returned to normal
sea water, they gain water in approximately the same amount and at the same
rate, so that they both become slightly larger, but keep approximately the same
size relative to each other (Photographs 15, 16, 17). The white halves never
attain the size of the white halves centrifuged off in normal sea water. If the
halves from the hypertonic sea water are placed in hypotonic sea water (60 per
cent), they still swell approximately the same amount, the red halves being in the
batch pictured a little larger than the whites (Photograph 18).
Size of nuclei in hvpo- and hvpertonic sea water and their recovery in sea water
(Table I and Plate I)
Though not directly related to the problem under discussion, the size of the
mature nucleus in hypo- and hypertonic sea water is of sufficient interest to be
recorded here. The nucleus of a normal mature unfertilized Arbacia egg is diffi-
cult to measure because it is imbedded in granules. However, when the egg is
centrifuged, the nucleus lies in the clear layer under the oil cap and can easily be
observed and measured in both the whole egg and the white half. The increase
in size in hypotonic sea water, and the decrease in hypertonic sea water is quite
apparent in photographs (Plate I). The nucleus of the normal egg in sea water
measures approximately 11.5 M in diameter; in 60 per cent sea water the diameter
is 16 /z, an increase to two and a half times its volume; in 80 per cent sea water the
diameter is 12.8 M; in 125 per cent sea water the diameter is 9.6 /*, a decrease to
about one half the volume of the normal nucleus (Table I A). The cell increases
to about one and one half its volume in 60 per cent, and decreases to three quarters
its volume in 125 per cent sea water. The percentage increase and decrease in
volume of the nucleus is greater than the percentage increase and decrease in
PLATE II
Photographs 13, 14. The two half-eggs obtained from centrifuging in hypotonic sea water
(60 per cent, as shown in Photographs 2, 3) after their return to normal sea water.
Photographs 15, 16, 17. The two half-eggs obtained from centrifuging in hypertonic sea
water (125 per cent, as shown in Photograph 12) after their return to normal sea water; the same
halves at 15-minute intervals. There was no further change in size in photographs taken several
hours later.
Photograph 18. The two half-eggs obtained from centrifuging in hypertonic sea water
(125 per cent, as shown in Photograph 12), after placing them in 60 per cent sea water.
Same magnification as in Plate I, approximately 275 X.
EGGS CENTRIFUGED IN ANISOTONIC SEA WATER
147
o
15
12. SO p.m.
60$ - 100$
125$ - 100$
17
l.p.m.
18
i
- 60$
PLATE II
148 ETHEL BROWNE HARVEY
volume of the egg. This may be due to changes in metabolism, or to a smaller
amount of osmotically inactive material in the nucleus, or it may be due to a
difference in the nuclear and cell membranes. Skowron and Skowron (1926) 3
have noted a similar volume difference of the germinal vesicle of the Sphaerechinus
granularis egg in comparison with the egg itself when treated with hypotonic solu-
tions. And Beck and Shapiro (1936) found the same thing true for the germinal
vesicle of the starfish egg swollen in 80 per cent sea water.
The nuclei of the whole Arbacia eggs returned to normal sea water after hypo-
and hypertonic sea water, regain their normal size (Table I B), but at a much
slower rate than the egg itself.
In the case of the germinal vesicle of the immature Arbacia egg, Churney
(1942) concluded that it swells and shrinks reversibly in anisotonic solutions, and
acts as a better osmometer than the egg itself. Beck and Shapiro (1936) have
likewise found that the germinal vesicle of the starfish egg shrinks and swells in
the same sense as the cell, and they have called attention to the fact that the rate
is slower for the nucleus than for the egg to attain equilibrium. The mature
nucleus of the Arbacia egg, therefore, seems in all respects similar to the immature
nucleus (germinal vesicle) of Arbacia and other sea urchins, and of the starfish,
with regard to swelling and shrinking. This is of interest and not necessarily to
be expected because (1) the membrane of the mature nucleus is a new formation
after the polar bodies are given off and (2) the contents of the mature and imma-
ture nucleus are different both in morphological structure (e.g. the nucleolus) and
in the amount of material present; the volume of the germinal vesicle of Arbacia is
about 50 times that of the mature nucleus.
Size of half -eggs obtained by centrifnging in normal sea water and then placing them
in hypo- and hypertonic sea water (Table I C)
The swelling of half-eggs obtained by centrifuging eggs in a 100 per cent sea
water-sugar medium and then placing them in hypotonic sea water has been
adequately studied by Lucke (1932b, 1940). He found that both the half-eggs
swelled in hypotonic sea water, but that the white half swelled a little more than
the red half because the latter contained more of the osmotically inactive material,
which he estimates as 12 per cent. His mean values are given in Table I D. My
figures agree fairly well with his. In Table I C, my figures are given for the
swelling and shrinking of the same eggs and half eggs as used in the other parts
of the same experiment (Table I A and B). One may thus compare, in the same
batch of eggs, the allocation of excess water in the two halves obtained by cen-
trifuging before and after treating with hypotonic sea water; and similarly for the
extraction of water in hypertonic sea water.
DISCUSSION
With a constant centrifugal force of 10,000 X g, Arbacia eggs break less
rapidly in hypotonic sea water and more rapidly in hypertonic sea water than they
do in normal sea water. The tension at the surface is increased with the increase
3 These authors found no decrease in size of the germinal vesicle in hypertonic glucose
(A = 2.57), though the cell shrank 57 per cent; this seems strange in view of their results for
hypotonic glucose.
EGGS CENTRIFUGED IN ANISOTONIC SEA WATER 149
of surface area (Cole, 1932), so that if this factor alone were considered, the eggs
should break less rapidly in hypotonic sea water, as they do. However, the
densities of the half-eggs in comparison with the medium must also be considered,
and these densities were not measured.
With regard to the size of the two halves, it is seen from the data presented
that when Arbacia eggs are kept in hypotonic sea water and centrifuged in a
similar medium, the egg breaks so that the light half is much larger than the heavy
half, whereas in normal sea water it is only slightly larger. The excess water is
distributed largely to the light half. Conversely, when the eggs are kept in
hypertonic sea water and centrifuged in a similar medium the egg breaks so that
the light half is usually slightly smaller than the heavy half. Much of the water
is taken away from the light half. This is perhaps what is to be expected since
it is the clear layer in the light half that contains most of the osmotically active
material. Similarly, the large white halves from the hypotonic sea water lose
much more water when returned to normal sea water than do the smaller, more
granular red halves. On the other hand, when the eggs are centrifuged in
hypertonic sea water, the granules are more evenly distributed between the two
halves, now nearly equal in size. The clear layer is small, the white half is quite
granular, and the granules in the heavy half are not well packed, so that there is
probably more liquid (osmotically active) material present among these granules
than is apparent to the eye. Thus, when these two halves are returned to normal
sea water, they swell approximately the same amount.
It might be of interest to compare the results obtained with hypo- and
hypertonic sea water with those previously obtained by changing the centrifugal
force (1941; compare Plate I of the present paper with Plate I of the previous
paper). A low force acts similarly to hypotonic sea water; the heavy granules
are well segregated, the light half is much larger than the heavy half, and the egg
breaks apart less readily. A high force acts similarly to hypertonic sea water; the
heavy granules are not well segregated, the light half is smaller than the heavy
half, and the egg breaks apart more readily. Perhaps one might conclude that
when the heavy granules are well packed, whether by using a low force or by
adding water, the granular half is smaller in comparison with the less granular,
and the egg breaks more slowly.
SUMMARY
1. Arbacia punctulata eggs, when centrifuged with a force of 10,000 X g, break
less readily in hypotonic sea water, and more readily in hypertonic sea water than
in normal sea water.
2. When broken apart in hypotonic sea water, the white half is very much
larger than the red half. The white half is much larger than the white half ob-
tained by centrifuging in normal sea water, the red half only slightly larger than
the red half obtained in normal sea water.
3. When broken apart in hypertonic sea water, the white and red halves are of
almost equal size, the white half usually a little smaller than the red half. When
broken apart in normal sea water, the white half is somewhat larger than the red
half. The white half from the hypertonic sea water is much smaller than the
white half from normal sea water, the red half nearly the same size.
150 ETHEL BROWNE HARVEY
4. When the halves obtained by centrifuging in hypotonic sea water are
returned to normal sea water, they both lose water, but the white half to a much
greater extent than the red half. They become of approximately the same size as
though they had been centrifuged in normal sea water.
5. When the halves obtained by centrifuging in hypertonic sea water are re-
turned to normal sea water, they both take up water to about the same extent.
The white half remains considerably smaller than when centrifuged in normal sea
water.
6. The nucleus of the mature unfertilized egg increases perceptibly in hypo-
tonic sea water and decreases in hypertonic sea water, to a greater percentage
volume than the egg itself. It attains normal size on return to sea water.
LITERATURE CITED
BECK, L. V., AND SHAPIRO, 1936. Permeability of germinal vesicle of the starfish egg to water.
Proc. Soc. Exp. Bid. and Med., 34: 170-172.
CHURNEY, L., 1942. Osmotic properties of the nucleus. Biol, Bull., 82: 52-67.
COLE, K. S., 1932. Surface forces of the Arbacia egg. Jour. Cell, and Comp. Physiol., 1: 1-9.
HARVEY, E. B., 1932. The development of half and quarter eggs of Arbacia punctulata and of
strongly centrifuged whole eggs. Biol. Bull., 62: 155-167.
HARVEY, E. B., 1936. Parthenogenetic merogony or cleavage without nuclei in Arbacia punctu-
lata. Biol. Bull, 71: 101-121.
HARVEY, E. B., 1940. A comparison of the development of nucleate and non-nucleate eggs of
Arbacia punctulata. Biol. Bull., 79: 166-187.
HARVEY, E. B., 1941. Relation of the size of "halves" of the Arbacia punctulata egg to cen-
trifugal force. Biol. Bull, 80: 354-362.
HARVEY, E. N., 1931. The tension at the surface of marine eggs, especially those of the sea urchin,
Arbacia. Biol. Bull., 61: 273-279.
LUCRE, B. AND CO-WORKERS.
McCuxcHEON, M., AND B. LUCRE, 1927. The kinetics of exosmosis of water from living
cells. Jour. Gen. Physiol., 10: 659-664.
McCuxcHEON, M., B. LUCKE, AND H. K. HARTLiNE, 1931a. The osmotic properties of
living cells (eggs of Arbacia punctulata). Jour. Gen. Physiol., 14: 393-403.
LUCKE, B., H. K. HARTLINE, AND M. MCCUTCHEON, 1931b. Further studies on the kinetics
of osmosis in living cells. Jour. Gen. Physiol., 14: 405-419.
LUCKE, B., AND M. McCuxcHEON, 1932a. The living cell as an osmotic system and its
permeability to water. Physiol. Rev., 12: 68-139.
LUCKE, B., 1932b. On osmotic behavior of living cell fragments. Jour. Cell, and Comp.
Physiol., 2: 193-199.
LUCKE, B., 1940. The living cell as an osmotic system and its permeability to water. Cold
Spring Harbor Symposia, 8: 123-132.
SKOWRON, S., AND H. SKOWRON, 1926. Les changements du rapport plasmonucleaire dans des
oeufs pas murs d'Oursins sous 1'influence de differences de la pression osmotique du
milieu. Bull, de VAcad. Polonaise des Sc. et des Lettr. Ser. B., 1926: 859-879.
THE SPERMATOZOON AND FERTILIZATION MEMBRANE OF
ARBACIA PUNCTULATA AS SHOWN BY THE
ELECTRON MICROSCOPE1
ETHEL BROWNE HARVEY AND THOMAS F. ANDERSON 2
(The Marine Biological Laboratory, Woods Hole; the Biological Laboratory, Princeton University;
and the Eldridge Reeves Johnson Foundation for Medical Physics, University of Pennsylvania)
Spermatozoa have been studied for many years with the light microscope,
and the general structure of many kinds of spermatozoa has been described.
This study of the spermatozoa of Arbacia punctulata was undertaken to throw
further light on their structure by the use of the electron microscope.
The fertilization membrane of the Arbacia egg which is thrown off two
minutes after fertilization is now generally believed to have been, at least in
part, the plasma (or cell) membrane before fertilization. The fertilization
membrane was therefore studied in the hope that the electron microscope would
throw some light on the structure of the plasma membrane.
TECHNIQUE
The preparation of various kinds of biological material for the electron
microscope has already been described in some detail (see Anderson, 1942, and
references given therein). Briefly the procedure involves: (1) the complete
removal of the sea water by washing several times with distilled water, in order
to avoid the formation of salt crystals; (2) placing the material on a thin collodion
membrane across a fine mesh wire screen (200 mesh per inch) ; (3) allowing it to
dry; and (4) placing the screen in the electron microscope. In the present work
an "RCA type B" microscope was used and the micrographs were taken with
60 kilovolt electrons.
The Arbacia sperm were taken directly from the testis of a freshly opened
animal and diluted in sea water. They were then mounted on the collodion
membrane to which they adhered, washed in several changes of distilled water,
and then dried.
The preparation of the fertilization membranes presented greater technical
difficulties; since they seemed to show no tendency to adhere to the collodion
membranes, they had to be freed from the eggs and washed before they could be
placed on the specimen screens. The fertilization membranes are formed about
two minutes after fertilization of the eggs in sea water at 23° C. It was found
that if the eggs are placed in distilled water one minute after formation of the
fertilization membranes, these rupture and the egg contents flow out, leaving the
empty membranes. If placed in distilled water a minute or two later, only part
of the contents come out, and still later none at all. The procedure of washing
1 We are indebted to the RCA for the use of their electron microscope at Woods Hole during
the summer of 1942.
2 Formerly RCA Fellow of the National Research Council.
151
152 E. B. HARVEY AND T. F. ANDERSON
the eggs several times in distilled water three minutes after fertilization was
therefore adopted for separating the membranes from egg material and freeing
them from salt. Under these conditions, the empty fertilization membranes
sometimes retain their spherical shape, but usually collapse and become crinkled;
they settle more slowly than the egg material to form a layer just above the
bottom of the dish where the eggs lie. With a micropipette, under a binocular
dissecting microscope, a number of membranes were taken up and deposited in
tiny drops at the centers of the collodion membranes. The specimens were
then dried in air and studied in the electron microscope.
RESULTS AND DISCUSSION
Arbacia spermatozoon
The Arbacia spermatozoon at high magnification with the light microscope
(Fig. 1) is observed to possess a pointed head with a flattened base adjacent to a
short, slightly narrower, middle piece which seems to contain a pair of spherical
bodies. The long thin filamentous tail extends from the middle piece. The
head (with middle piece) measures approximately 4 ju long and 2 n across the
base; the tail is approximately 45 n long. When placed in distilled water, the
heads were observed to swell to about twice their original size.
In the electron microscope, the changes in structure caused by washing and
drying are immediately apparent (Figs. 2 to 4). In most cases (except Figure 3),
the heads have lost their characteristic arrow-head shape, and material appears
to be flowing out of them. There is no distinct middle piece. The tails are, in
most cases, coiled around the heads and consist of strands; the ends resemble
frayed ends of rope unwrapped into separate strands (Fig. 2). The strands
themselves are frequently detached, broken up, and strewn about the field
(Fig. 3).
When examined more closely under higher magnification (Figs. 3, 4), a
number of interesting features are apparent in the tails. Each tail appears to
have been made up of about ten strands of uniform thickness, each having a
diameter of about 50 m/x. In some of the micrographs, the tail has the appearance
of a thick core surrounded by a sheath, but this appearance might be produced
by a number of fibrils being superimposed at the center and flanked by one or
two single fibrils. Occasionally one sees individual fibrils apparently broken up
into short rods lined up in a row (Fig. 3), but this may be an artifact produced
by drying, shrinking, and breaking. The regularly spaced cross striations which
appear along the tail in certain areas (Fig. 4) may be characteristic of the material
as has been reported for collagen fibers (Schmitt, Hall and Jakus, 1942) or may
be an artifact of drying analogous to the formation of the rods noted above,
but on a smaller scale.
PLATE I
FIGURE 1. Living spermatozoa of Arbacia punctulata as photographed with the light micro-
scope. X 1,000.
FIGURE 2. Spermatozoa of Arbacia punctulata micrographed with the electron microscope
showing the appearance after washing in distilled water and drying. X 2,200.
FIGURE 3. Head and fragments of the tail of a spermatozoon at high magnification with
the electron microscope. X 15,000.
ARBACIA SPERM AND FERTILIZATION MEMBRANE
153
PLATE I
154 E. B. HARVEY AND T. F. ANDERSON
Unfortunately, the heads are too thick to show much internal structure. In
some of the micrographs, one sees a small round area of low density which might
represent a vacuole. There is also, in one of the micrographs (Fig. 4), a lighter
area of the head having the appearance of a membrane. This is interpreted as
the outer membrane left more or less intact on drying while the material inside
has withdrawn and flowed out at the sides. It is not possible to determine the
structure of the nuclear material from these micrographs. Some of the material
found in the neighborhood of the heads appears to have interesting structure,
such as the small rings, but it is impossible to identify it at this time.
Fertilization membrane of Arbacia
In the light microscope, the fertilization membrane of Arbacia punctulata
appears as a uniformly thin and transparent membrane 3 to 5 M from the surface
of the egg. It is quite elastic when first formed, as shown by the fact that in
high centrifugal fields it stretches from a sphere having a diameter of 80 /x to a
spheroid having a length of 140 ^ (Harvey, 1933, and unpublished observations).
Five minutes after fertilization, however, the membrane thickens and hardens
and resists stretching. Membranes freed from the eggs one minute after fertil-
ization in distilled water have been observed to last 12 hours without any apparent
change.
A number of electron micrographs of various fertilization membranes were
taken and none showed anything but a thin amorphous structure (the membrane)
sprinkled with what appears to be debris (Fig. 5). This debris may actually
represent the structure of certain components of the cell or plasma membrane
of the unfertilized egg, but the fact that they are neither characteristic in shape
nor distributed in definite patterns on the surface prohibits one from attaching
any special significance to them. There are no pores of sufficient size to be
recognizable as such in the micrographs. From the apparent density of the
micrograph one can estimate the thickness of the fertilization membrane, when
first formed and dried, to be of the order of 25 HIM- It is of interest to note
that this estimate is approximately the same as that of the membrane of the red
blood cell. In the recent work of Zwickau (1941), who studied the red cell
membranes with an electron microscope, the thickness of the membrane of the
dried ghost is given as 20-30 mju. Other estimates of the thickness of the intact
red blood cell membrane, including water and diffusible proteins range from
20 m/z to as much as 50 m^u (see Ponder, 1942). The electron micrographs of
the red blood cell membranes given by Zwickau show no definite structures.
PLATE II
FIGURE 4. Spermatozoon, disrupted by distilled water, showing the multiple stranded
structure of the tail with cross striations, and the remains of what may have been the membrane
of the head — with the electron microscope. X 10,000.
FIGURE 5. Electron micrograph of the fertilization membrane of an Arbacia punctulata
egg. At the top of the field is the collodion film on which the specimen is mounted with a hole
in it at the upper left hand corner. The fertilization membrane comes up from the bottom of
the field and folds over on itself near the top. The dark line extending from the upper left hand
corner is a wrinkle in the film. Note the frayed edge of the fertilization membrane to the left
of the middle of this wrinkle. X 22,000.
ARBACIA SPERM AND FERTILIZATION MEMBRANE
155
5
PLATE II
156 E. B. HARVEY AND T. F. ANDERSON
SUMMARY
1. As studied with the electron microscope, the tail of the Arbacia punctulata
spermatozoon is found to disrupt into about ten distinct fibrils when it is washed
in distilled water and dried. Each fibril is about 50 m/j. in thickness. Regularly-
spaced cross striations also appear in the tail structure, but these may be produced
in the washing and drying process.
2. A method of obtaining the fertilization membranes of Arbacia punctulata
eggs free from egg material is described. When these were washed in distilled
water and dried for examination, the electron microscope revealed no regular
structures nor definite patterns. The thickness of the fertilization membrane,
when first formed and dried is estimated to be of the order of 25 m/z.
LITERATURE CITED
ANDERSON, T. F., 1942. The application of the electron microscope to biology. The Collecting
Net, 17: 4-6.
HARVEY, E. B., 1933. Effects of centrifugal force on fertilized eggs of Arbacia punctulata as
observed with the centrifuge-microscope. Biol. Bull., 65: 389-396.
PONDER, E., 1942. Quantitative aspects of the disc-sphere transformation produced by lecithin.
Jour. Exp. Biol., 19: 220-231.
SCHMITT, F. O., C. E. HALL, AND M. A. JAKUS, 1942. Electron microscope investigations of
the structure of collagen. Jour. Cell, and Conip. Physio!., 20: 11-33.
ZWICKAU, R., 1941. Zur Frage d. Erythrocytenmembrane. Inaug. Diss. From the Lab. f. Uber-
microscopie d. Siemens u. Halske, Berlin.
THE DEVELOPMENT OF AN ENZYME (TYROSINASE) IN THE
PARTHENOGENETIC EGG OF THE GRASSHOPPER,
MELANOPLUS DIFFERENTIALS *
JOSEPH HALL BODINE AND THEODORE NEWTON TAHMISIAN
(Zoological Laboratory, The State University of Iowa, Iowa City)
INTRODUCTION
Parthenogenesis has offered opportunities for investigating problems in almost
every phase of experimental biology. In the grasshopper (Melanoplus differ-
entialis] parthenogenesis has been studied from the developmental and cytological
aspects by King and Slifer (1934). Studies on protyrosinase formation and
activation in the normal fertilized grasshopper egg have been carried out in some
detail in this laboratory and it, therefore, becomes of interest to compare such
results with those from parthenogenetic eggs.
MATERIALS AND METHODS
Female grasshopper nymphs (Melanoplus differentialis) in the third instar
were segregated and raised free of males. At maturity their eggs were collected
daily and kept in filter paper on moist sand at 25° C. These eggs were prepared
for experimentation in the following manner: Approximately 150 eggs of a known
chronological age and temperature history were placed in 0.9 per cent NaCl solu-
tion and each egg was scraped free of its chorion at the posterior end in order to
determine the presence of a cuticle. Only those eggs with a cuticle were chosen.
These in turn were sterilized in 70 per cent ethyl alcohol for 10 minutes. Ten
to 25 eggs were taken from this lot and dissected to determine the presence of an
embryo as well as its morphological age (Slifer, 1932). Of those remaining 100
were taken and placed in a glass mortar, rinsed with redistilled water, and then
triturated with 0.9 per cent NaCl. The triturate was diluted to 10 ml. and
centrifuged at 1,500 times G. for 10 minutes. The lipoidal A layer and the shell
fragments constituting the major portion of the C layer (Bodine and Allen, 1938)
were discarded since practically no protyrosinase is present in them. The
protyrosinase content of the B layer was determined manometrically.
Each vessel of the Warburg manometer contained 1 ml. of the enzyme extract,
0.5 ml. of Sorensen's phosphate buffer (0.2 M. in respect to the phosphate) at
pH 6.8, 0.3 ml. of 1 per cent aerosol OT solution, 0.9 per cent NaCl solution, and
0.3 ml. of a 0.4 per cent solution of tyramine-HCl in the side bulb. Ten minutes
after equilibration at 25° C. the substrate in the side bulb was decanted into the
main chamber of the vessel and the first reading taken two minutes after mixing.
The manometers were shaken at 120 oscillations per minute through an amplitude
of 2 cm.
* Aided by grant from the Rockefeller Foundation for research in cellular physiology.
157
158 J. H. BODINE AND T. N. TAHMISIAN
The morphological age of the embryos in the diapause eggs was determined
by removing the chorion and noting the position of the eye pigment. At diapause
the head of the embryo is at the posterior end of the egg. After diapause the head
of the embryo faces the anterior end of the egg making the determination of the
morphological stage relatively simple (Slifer, 1932). Only those eggs which, in
diapause, had a cuticle and an embryo were chosen for experiments. Such selec-
tions were especially necessary for parthenogenetic eggs to insure reproducible
results.
Five day old parthenogenetic eggs were divided into two groups; one was kept
as control and the other was irradiated at 1,000 r (Ray, 1938; Bodine and Allen,
1941). Both groups were then kept at 25° C. Daily determinations of the
protyrosinase content were made starting with the tenth day after irradiation.
Fertilized eggs collected in the usual manner were allowed to develop at 25° C.
and on the tenth day divided into two groups. One group was placed at 0° C.
and the other was kept at 25° C. to serve as control. A second group of eggs was
divided into two groups on the fifteenth day of prediapause development at 25° C.
One group was left as a control at 25° C. while the other was placed at 0° C. A
third group was divided on the fifteenth day of prediapause development at 25° C.
Those placed at 0° C. were further divided after the tenth day and one lot of these
was then placed at 25° C. Daily determinations of the protyrosinase content of
the eggs were made.
Prediapause and diapause fertilized eggs were divided into two groups. One
lot from each of these groups was kept as a control at 25° C. The other groups
were separately placed into glass bottles and sealed. The eggs in the sealed
bottles were then subjected for one hour to — 78° C. with the aid of dry ice and
ether. The protyrosinase content of these four groups was determined immedi-
ately after the experimentals were so treated. Thereafter they were all placed at
25° C. and daily determinations of the protyrosinase content of the cold treated
and control eggs were made.
RESULTS
During prediapause from the day of laying until the fifteenth day of develop-
ment at 25° C. parthenogenetic eggs contain no detectable protyrosinase (Fig. 1).
Enzymogenesis begins on approximately the fifteenth day of development and the
enzyme increases in amount until the twenty-fifth day. At diapause the
protyrosinase content of the parthenogenetic egg remains constant at the level
attained on the twenty-fifth day. Unlike parthenogenetic eggs the protyrosinase
of the normal fertilized egg appears much earlier, namely on the eighth day of
development (Fig. 1), (Bodine and Boell, 1935). It gradually increases in amount
until at diapause the protyrosinase content is at a maximum (Bodine and Allen,
1941). It is obvious that there is a marked lag in the appearance of protyrosinase
in the parthenogenetic egg. King and Slifer (1934) described a lag in the morpho-
logical, cytological and developmental aspects of the parthenogenetic grasshopper
egg-
Ray (1938) observed that irradiation of normal fertilized eggs with 1,000 r on
the fifth day of development destroyed the embryo but did not affect the formation
of protyrosinase. Later it was noted (Bodine and Allen, 1941) that irradiation
ENZYMES IN PARTHENOGENESIS
159
with 1,000 r on the first day of development destroyed both the embryo and the
serosa cells and that no protyrosinase was formed. Irradiation with 1,000 r on
the fifth day after laying, however, was without effect on the function of the serosa
cells in their formation of the yellow cuticle, white cuticle, and protyrosinase. It
became of some interest, therefore, to compare the effect of a similar dose of
x-irradiation on five day old parthenogenetic eggs. Since all parthenogenetic eggs
do not develop (King and Slifer, 1934) it was necessary to compare the number of
eggs that formed cuticle several days after irradiation with non-irradiated control
parthenogenetic eggs. In both cases approximately 70 per cent formed cuticles.
The presence of cuticle is important as an index of the functional state of the
serosa which also seems to produce most of the protyrosinase (Bodine and Allen,
1941). In parthenogenetic eggs the formation of the cuticle begins approximately
on the tenth day of development. The protyrosinase content between the
fifteenth and twenty-fifth day of development was similar in irradiated and non-
100-
75-
»0
x 50-
0-
8 10
15
20
25
DAYS
30
35
40
45
FIGURE 1. Shows amounts of enzyme in normal fertilized eggs and parthenogenetic eggs.
Abscissae, developmental time in days at 25° C. Ordinate, reciprocal half oxidation period X 103.
O = normal fertilized eggs. • = parthenogenetic eggs. X = irradiated parthenogenetic eggs.
irradiated parthenogenetic eggs (Fig. 1). It should be pointed out that in this
case also the amount of protyrosinase at diapause is half as much as in the normal
fertilized irradiated eggs (Ray, 1938; Bodine and Allen, 1941).
In order to check certain factors such as lag in development, high mortality,
etc., possibly related to the formation of a reduced amount of enzyme in the
parthenogenetic egg, the following experiments were carried out with fertilized
eggs.
Fertilized eggs placed at 0° C. immediately after laying did not develop. Over
a period of eighteen days no protyrosinase was detectable. When eggs so treated
were placed at 25° C. protyrosinase appeared on the twenty-fifth day after laying.
It should be pointed out that these eggs in reality were at a developmental temper-
ature for 7 days ± 1 day (Fig. 2). The amount of protyrosinase and the de-
velopment of the embryos were similar to control eggs kept for a similar period at
developmental temperatures. Eggs placed at 0° C. on the fifteenth day of
development also remained at this developmental stage. No change in the
160
J. H. BODINE AND T. N. TAHMISIAN
protyrosinase content over that found on the fifteenth day was detectable. When
these eggs were returned to 25° C. development and protyrosinase content in-
creased in a normal fashion (Fig. 2). The ultimate protyrosinase content of these
eggs was similar to that found in normal ones regardless of the stage or the length
of time they were inhibited.
Normal fertilized diapause eggs killed by subjection to - - 78° C. show a slight
drop in enzyme content immediately after freezing (Bodine and Allen, 1941).
Thereafter the amount of enzyme does not change significantly for a period of 18
days at 25° C. On the other hand the protyrosinase content of the normal
prediapause fertilized egg remains constant for a period of 18 days after subjection
to the low temperature (Fig. 3).
100-
75-
o
X
50-
25-
0 -
T~ ~l~ ~r~ -T- — r-
10 15 20 25 30
DAYS
35 40
FIGURE 2. Shows effect of developmental block, due to low temperature, on the enzyme
content of fertilized eggs. Ordinates as in Figure 1. Abscissae, time in days. Arrows indicate
period of exposure to 0° C. A = control eggs at 25° C. • = put at 0° on tenth day; on
eighteenth day put at 25° C. O = put at 0° C. on fifteenth day. D = taken from 0° C. on
the twenty-fifth day and put at 25° C.
DISCUSSION
In parthenogenetic eggs the lag in protyrosinase formation is doubtless due
to the lag in the developmental aspects of these eggs (King and Slifer, 1934). The
lesser amount of the protyrosinase ultimately produced in the parthenogenetic egg
is a matter'for some speculation.
In the course of parthenogenetic development, several anomalies occur, e.g.,
haploidy, retarded growth, undifferentiated growth of the embryonic cells, and a
high rate of mortality (King and Slifer, 1934). Concerning a haploid condition,
King and Slifer suggest that a total haploid condition in all probability does not
permit the embryo to develop. On the other hand they have observed partial
haploidy in individual embryos. They believe that in order for an embryo to de-
velop certain of its cells essential for the propagation of the embryo, must become
diploid while others may remain in a haploid condition. Haploid, diploid and
triploid sets of chromosomes in individual embryos have been observed in the
present work. Concerning the early differentiation of the serosa it may well be
ENZYMES IN PARTHENOGENESIS
161
that these nuclei in the parthenogenetic egg originate from haploid cells. Since
the serosa plays such an important part in the formation of protyrosinase (Bodine
and Allen, 1941) it is probable that a haploid condition may produce half the
amount of protyrosinase in comparison to the normal diploid egg. We were
unable to observe chromosomes in the serosa nuclei after they were morphologically
differentiated. Under normal conditions the serosa nuclei in fertilized eggs in-
crease in size and chromatin content by a peculiar type of endomitosis (Tahmisian,
Allen, and Bodine, 1942). This type of growth of the serosa nuclei was also ob-
served in the parthenogenetic eggs. As far as we can determine, normal differ-
entiated serosa cells in general do not deviate morphologically from those found
in the parthenogenetic eggs. In one case only a serosa from a parthenogenetic
egg had many small nuclei interposed with normal appearing large ones.
In order to ascertain the effect of retardation in the development of normal
fertilized eggs on the formation of protyrosinase, several lots of eggs were retarded
100-
75-
50-
25-
10
DAYS
15
20
FIGURE 3. Shows effect of killing fertilized eggs by subjection to — 78° C. on their enzyme
content. Ordinates and abscissae as in Figure 2. O = control diapause eggs. • = diapause
eggs subjected to — 78° C. A = enzyme content of 15-day eggs. D = 15-day eggs subjected
to - 78° C.
in development by subjection to 0° C. The results of these experiments have
already been mentioned above (Fig. 2). No matter at what stage the eggs were
experimentally retarded in relation to the time sequence of development, the
maximum amount of enzyme ultimately formed is equivalent to the amount of
protyrosinase found in the control eggs. We may, therefore, conclude that the
reduced amount of protyrosinase in the parthenogenetic eggs can not be accounted
for on the basis that at a previous time they had been retarded in their develop-
ment. Since each of the parthenogenetic eggs selected contained an embryo and
a cuticle the effect of undifferentiated growth may be ruled out.
King and Slifer (1934) also pointed out that many of the parthenogenetic eggs
die during development. In spite of the fact that the parthenogenetic eggs were
selected each day before analysis for protyrosinase it was possible that the results
were due to the presence of eggs that apparently were normal but which really
were dead. In order to determine the effect of killing the eggs on their protyrosinase
content, two lots killed by subjection to - - 78° C. were analyzed daily. It is ap-
parent (Fig. 3) that no detectable change in enzyme content was noted in dead
162 J. H. BODINE AND T. N. TAHMISIAN
prediapause eggs for a period of 18 days. On the other hand there is a very small
drop in the protyrosinase content of the killed diapause egg. As far as we can
determine the parthenogenetic eggs used in all experiments were not dead. An
egg, dead for 24 hours or more, changes color from a buff to dark sepia or black
which in no case was observed in the parthenogenetic eggs used. We, therefore,
infer that the protyrosinase content of the egg that has developed to a definite
morphological age though dead contains the same amount of enzyme as a normal
egg of the same age.
Another question which at present cannot be answered is suggested by the fact
that all parthenogenetic eggs develop into females (King and Slifer, 1934). Is
the protyrosinase content of normal fertilized female eggs one third that of the
protyrosinase content of the males? If so, then a sample containing equal repre-
sentation of eggs that will develop into males and females would of necessity have
two times as much protyrosinase as do the eggs that will develop into females.
And since all of the parthenogenetic eggs develop into females (King and Slifer,
1934), the presence of half the amount of protyrosinase in these eggs might, there-
fore, be accounted for on this basis.
The fact that the parthenogenetic eggs in practically all cases have exactly
half as much protyrosinase as compared to the normal fertilized ones suggests that
the male element donates some peculiar capacity to the normal developing egg to
form twice as much protyrosinase as can be produced by the female element alone.
Another possibility is that the unfertilized egg though appearing to be well
coordinated for morphological differentiation is physiologically not well coordi-
nated. It would be of interest to see if parthenogenetic eggs removed by another
generation would contain still less protyrosinase.
SUMMARY AND CONCLUSIONS
1. Development and rate of growth of the enzyme tyrosinase have been
studied in the parthenogenetic egg of the grasshopper, Melanoplus differ entialis.
2. A marked lag in the appearance of the enzyme in the parthenogenetic egg
occurs.
3. Total amount of enzyme found in the parthenogenetic egg is approximately
50 per cent of that found in the normal fertilized egg.
4. Parthenogenetic eggs subjected to x-irradiation on the fifth day of de-
velopment show no change in the amount and rate of production of the enzyme-
suggesting, as in normal eggs, the production of the enzyme by the serosa cells.
5. Results of experiments are presented which tend to show that arrested de-
velopment, or killing of eggs, by low temperature do not produce lowered amounts
of enzyme in eggs thus treated.
6. Possible explanations for the production by parthenogenetic eggs of lowered
amounts of enzyme are given.
LITERATURE CITED
BODINE, J. H., AND E. BOELL, 1935. Enzymes in ontogenesis (Orthoptera). I. Tyrosinase.
Jour. Cell, and Comp. Physiol., 6: 263-275.
BODINE, J. H., AND T. H. ALLEN, 1938. Enzymes in ontogenesis (Orthoptera). IV. Natural
and artificial conditions governing the action of tyrosinase. Jour. Cell, and Comp.
Physiol., 11: 409-423.
ENZYMES IN PARTHENOGENESIS 163
BODINE, J. H.( AND T. H. ALLEN, 1941. Enzymes in ontogenesis (Orthoptera). XX. The site
of origin and the distribution of protyrosinase in the developing egg of a grasshopper.
Jour. Exp. Zool., 88: 343-352.
KING, R. L., AND E. SLIFER, 1934. Insect development. Maturation and early development of
unfertilized grasshopper eggs. Jour. Morph., 56: 603-619.
RA\, O. M., 1938. Effects of roentgen rays on the activation and production of the enzyme
tyrosinase in the insect egg (Orthoptera). Radiology, 31: 428-437.
SLIFER, E., 1932. Insect development. IV. External morphology of grasshopper embryos of
known age and with a known temperature history. Jour. Morph., 53: 1-21.
TAHMISIAN, T. N., T. H. ALLEN, AND J. H. BODINE, 1942. Endomitosis (?) in grasshopper serosa
cells. Anal. Rec. (Abstr.), 84: 502-503.
A QUANTITATIVE STUDY OF ANAPHASE MOVEMENT IN THE
APHID TAMALIA
HANS RIS
(Department of Biology, The Johns Hopkins University, Baltimore, and
the Marine Biological Laboratory, Woods Hole]
No single phase of mitosis has been discussed as often as the anaphase move-
ment of chromosomes. The precision of the movement, the relatively large
distances covered and the possibility of correlation with definite cellular structures
make it better suited for causal analysis than any other phase of cell division. A
great number of ingenious hypotheses have been designed to account for the
movement of chromosomes, making use of practically every known chemical and
physical process which could bring chromosomes from the metaphase plate to the
poles. But so far none has been satisfactory and none has been verified even
partly by experiment. To some extent this failure is due to the difficulty of the
subject. Another reason is the over-emphasis on deductive schemes which may
explain a movement of bodies like chromosomes but which are without empirical
foundation. This was clearly stated by Belar (1929a) when he pointed out that
we have to find out how the chromosomes move before we can ask what forces are
responsible for this movement. What is needed then is a quantitative description
of the chromosome movement derived from the study of living cells in division.
There are in the literature only two such accounts: one by Belar (1929a) in
spermatocytes of the grasshopper (Chorthippus) and the other by Barber (1939)
in Tradescantia staminal hair cells. Belar derived his data from measurements
on photographs which were taken at intervals of several minutes. This can give
only a very rough picture of the chromosome movement. Barber measured the
distance between disjoining kinetochores, again on photographs, at intervals of
one-half or one minute and therefore could offer a more complete description of
the anaphase movement. However, the position of the long chromosomes in the
metaphase plate and in early anaphase make exact measurements in these stages
almost impossible. The present investigation was undertaken to provide more
data on the movement of chromosomes in living cells as a basis for both experi-
mental attacks and theoretical interpretations.
MATERIAL AND METHODS
The bearberry aphid Tamalia coweni was found to be favorable material for
the study of cell division in both spermatocytes and embryonic cells. Several
males or parthenogenetic females are dissected in a drop of paraffin oil on a
coverglass. The testes — or young embryos — come to lie in a small pool of body
fluid surrounded by paraffin oil. The coverglass is then inverted over a de-
pression slide. Cells have thus been kept alive and normally dividing for more
than 10 hours. A glass container with ferrous ammonium sulphate between lamp
164
ANAPHASE MOVEMENT OF CHROMOSOMES 165
and microscope prevented any heating due to the light source. The temperature
varied from 22° to 26°. A good indication of the normality of conditions is given
by the close agreement of the curves of different cells from different individuals
(Fig. la and 4a). In addition spermatogonia and spermatocytes of Protenor
belfragii and Thelia bimaculata were studied in a hanging drop of paraffin oil.
To analyze the movement of the chromosomes, a metaphase plate in side view
is selected and with beginning anaphase the distance between the kinetochores of
the daughter chromosomes recorded at intervals of one half to one minute with a
camera lucida. This method was found to be simpler and more accurate th^n
measurements on photographs. The error as determined from 20 measurements
is ± 4 per cent. The various distances are then calculated in micra and plotted
against time (Barber, 1939). We thus get a curve describing the movement of
the chromosomes.
All forms studied here are characterized by a diffuse spindle attachment and
therefore parallel disjunction. (Hughes-Schrader and Ris, 1941; Ris, 1942).
This makes it easier to follow one single chromosome from metaphase to telophase.
To avoid the error due to the curvature of the spindle a chromosome near the
spindle axis is chosen. As a complement to the studies on live cells fixed and
stained sections were used to measure the length of chromosomal fibers as well as
the whole spindle with increasing separation of the daughter chromosomes.
The optics used consisted of a 2 mm. Zeiss oil immersion N.A. 1.4 and
15 X ocular.
Anaphase movement in secondary spermatocytes of Tamalia
The type of anaphase movement characteristic for the forms studied is most
clearly shown in the secondary spermatocytes of Tamalia (Fig. la). When the
daughter chromosomes begin to separate they are first connected by a "gray"
mass which then breaks up into a few strands. These probably are identical with
the Feulgen positive chromosome connections found in fixed cells (Ris, 1942). In
a frontal or end view the chromosomes have a very characteristic dumb bell shape.
The movement of the chromosomes is slow until all these connections have dis-
appeared. Now it increases in speed and remains nearly uniform for several
minutes, when it comes to a halt for about two minutes. The motion is then
resumed only to slow down once more as the end of anaphase is approached. The
second movement after the plateau in the curve coincides with the elongation of
the cell. Previously the cell is spherical or in rare cases has elongated only
slightly. Within about ten seconds after the beginning of elongation the cleavage
furrow appears (arrow in Fig. la).
How can the interruption in the movement of the chromosomes be explained?
The coincidence of cell elongation and the second movement of the chromosomes
suggests that both may be connected with a stretching of the spindle. We could
then picture the anaphase movement as composed of two phases: in the first the
chromosomes approach the poles, or in other words, the chromosomal fibers
shorten.1 In the second phase the spindle stretches and moves the chromosomes
farther apart. To prove this hypothesis we must take recourse to stained sections
1 Since nothing is known about the mode of action of chromosomal fibers the term "shortening
of chromosomal fibers" is used throughout this paper.
166
HANS RIS
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ANAPHASE MOVEMENT OF CHROMOSOMES
167
where we can measure the length of chromosomal fibers and spindle for various
distances between the daughter chromosomes. Such measurements are plotted
in Figure Ib. They show clearly that in the first part of the movement the
chromosomal fibers shorten while the spindle remains constant in length. In the
second phase the chromosomal fibers remain constant while the spindle begins to
stretch, causing the further movement of the chromosomes. Making allowance
TAMALIA EMBRYONIC MITOSIS
10-
to
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FIG.2
LENGTH OF CELL
•TA40
TA40
• TA24
TAI8
/ ,—• TA25
7 /
J\ /-I
7
23456 789 10
| TIME IN MINUTES
16
15
14
13
UJ
u
I
-12 o
z
LJ
-II
-10
FIGURE 2. Chromosome movement in embryonic cells of Tamalia. Measurements on living
cells. For Ta 40 both distance between kinetochores and length of cell are plotted.
for shrinkage at fixation, Figures la and Ib can be compared. Shrinkage was
calculated by comparing the maximum separation of daughter chromosomes in
living and fixed cells and results in a shortening of the interchromosomal distance
by one-fourth. In the living cell the break in the curve occurs when the daughter
chromosomes are from 7 to 8 /j. apart, in the fixed cells accordingly at a separation
of 5 to 6 IJL. It is also interesting that the elongation of the cell corresponds closely
to the increase of spindle length (increase in length of cell 4 n, of fixed spindle 3 ju).
168
HANS RIS
The movement of the chromosomes in this division can now be described in
the following way: first slowly, then faster the chromosomes approach the poles
apparently through the action of the chromosomal fibers. When they are from
7 to 8 n apart this movement ceases and for a short time the chromosomes come
to rest. Then the spindle begins to elongate, causing the final separation of the
chromosomes. The distance from chromosomes to poles remains constant in this
latter phase.
Anaphase movement in embryonic cells of Tamalia
Young embryos dissected from parthenogenetic females have many somatic
cells in division. Curves for the anaphase movement are obtained as in
spermatocytes. As there are many different types of cells of various sizes the
curves differ quantitatively. The character of the movement, however, is the
same in all cells and identical with that in secondary spermatocytes (Fig. 2).
There is the initial slow movement, the first fast movement, the pause and the
second movement coinciding with cell elongation. Because of the difference of
FIGURE 3. Anaphase in embryonic cell of Tamalia. Penetration of cytoplasmic
granules in between the daughter plates. See text.
the cells a comparison with measurements of fixed material is impossible. Yet the
curves agree well enough with those of secondary spermatocytes to justify the
conclusion that the nature of the movement is the same. The velocity of the
chromosomes is greater than in spermatocytes and large enough so that the
chromosomes can actually be seen in motion under the microscope.
The observation of these cells during anaphase furnishes some interesting data
on the spindle. The cytoplasm contains a great number of dark granules of
various sizes. When the spindle is formed at metaphase they accumulate along
its surface and thus outline its shape. In constant Brownian movement they can
be seen bouncing off the surface of the spindle, but never penetrating it. Towards
the end of metaphase the majority of granules has accumulated around the
equatorial region of the spindle. In the first part of anaphase the spindle retains
its characteristic shape, outlined by the cytoplasmic granules. As soon as the cell
begins to elongate, indicating the stretching of the spindle, the granule-free region
between the daughter plates becomes constricted in the middle and shaped like an
hour glass. Soon afterwards cytoplasmic granules rush into the midregion of the
spindle, continuously in unrestricted Brownian movement (Fig. 3).
ANAPHASE MOVEMENT OF CHROMOSOMES
169
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ANAPHASE MOVEMENT OF CHROMOSOMES
171
Anaphase movement in primary spermatocytes of Tamalia
The first spermatocyte division of the aphid is unusual in several ways. The
univalent X chromosome is stretched into a flat sheet at anaphase and passes
undivided into the larger of the unequal daughter cells (cf. Ris, 1942).
The anaphase movement also is different from that in cells previously de-
scribed (Fig. 4a). The chromosomes very soon reach their maximum velocity
and then gradually slow down towards the end of anaphase. The curve resembles
the second movement in secondary spermatocytes, which was found to be caused
by spindle elongation. Indeed the measurements of chromosomal fibers and
spindle in fixed cells show that the entire movement of the chromosomes is due to
the stretching of the spindle. The chromosomal fibers remain constant in length,
THELIA MEIOSIS I
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3-
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FIG.6
10 15 20
TIME IN MINUTES
25
30
FIGURE 6. Chromosome movement in a primary spermatocyte of Thelia.
i.e., the chromosorrfes do not get nearer the poles (Fig. 4b). The arrow in
Figure 4a marks the appearance of the cleavage furrow.
Is this kind of anaphase characteristic for primary spermatocytes or is it
peculiar to the aphid? To answer this question the anaphase movement in pri-
mary spermatocytes of the hemipteran Protenor and the homopteran Thelia was
analyzed.
Anaphase movement in primary spermatocytes of Protenor and Thelia
The chromosome movement in a primary spermatocyte of Protenor is shown in
Figure 5a. The curve for the autosomes is of the same type as those found for
somatic mitosis and secondary spermatocytes in the aphid. Again the cleavage
furrow appears shortly after the second movement has started. Measurements of
172
HANS RIS
fixed cells finally show that anaphase here too consists of the two phases, the
approach to the poles and the spindle elongation.
Interesting is the behavior of the univalent X chromosome. In the first
meiotic division it splits equationally but the daughter chromosomes lag behind
the autosomes (Schrader, 1935). What is the reason for this delay? The curve
for the X chromosome in Figure 5a shows that it is the first part of anaphase which
differs from that of the autosomes. Chromosomal fibers are present (Schrader,
1935), but if they are responsible for the movement towards the poles, they are in
some way hampered in their function. In the second phase of the movement,
which is related to the stretching of the spindle, the X chromosome behaves like
the autosomes and even partially catches up with them.
PROTENOR SPERMATOGONIA
P. 5
5 10 15
TIME IN MINUTES
FIGURE 7. Chromosome movement in two spermatogonia of Protenor.
The first meiotic anaphase of Thelia is similar in character to that of Prptenor
(Fig. 6) and thus also of the same type as found in somatic cells and secondary
spermatocytes of the aphid. It must be concluded, therefore, that the anaphase
movement of the first meiotic division in Tamalia is different from that in Protenor
and Thelia and represents an exceptional case.
Anaphase movement in spermatogonia of Protenor
In Figure 7 the anaphase movement in two spermatogonia of Protenor is re-
corded. In P4 the distance between the ends, in P5 that between the middle of
two daughter chromosomes was measured. A comparison of the two curves
ANAPHASE MOVEMENT OF CHROMOSOMES
173
shows how the ends of the chromosomes separate first while the midregion lags
until the daughter chromosomes are fully separated (+ in P6). Again the move-
ment consists of two phases, separated by a short pause.
DISCUSSION
The measurements on living cells have furnished curves which describe in
detail the movement of the chromosomes at anaphase. In the cells studied it
TABLE I
Anaphase movement in secondary spermatocytes and embryonic cells of Tamalia.
d.k. = distance between kinetochores; I.e. = length of cell
Spermatocytes II
Embryonic cells
Ta34
Ta 40
Time
(minutes)
Ta26
25° C.
d.k.GO
Ta 27
24° C.
d.k.OO
Ta 29
24° C.
d.k.GO
23° C.
Ta 18
24° C.
d.k.GO
Ta 24
26° C.
d.k.(/i)
Ta-25
25° C.
d.k.GO
22° C.
d.k.GO
I.e. GO
d.k.GO
l.c.GO
0
1.8
2.0
2.0
2.2
11.7
2.0
1.6
1.6
1.4
11.2
1
2
2.0
—
2.5
—
—
2.5
2.0
1.8
1.8
11.2
1
2.5
2.2
2.7
—
—
2.7
2.7
1.8
2.7
11.2
1*
2.5
2.7
2.7
—
—
3.2
4.0
2.0
4.7
11.2
2
2.7
2.9
2.7
2.7
11.7
4.3
5.6
2.0
5.2
11.4
2i
2.7
3.8
3.1
3.6
12.1
4.5
6.7
2.2
6.3
14.0
3
3.6
4.0
3.1
3.8
12.6
4.5
6.7
2.7
6.9
14.4
3*
3.8
4.5
3.3
—
—
5.2
7.4
—
7.6
14.8
4
4.5
5.2
4.5
4.5
12.6
—
7.6
4.0
7.9
14.8
4*
4.9
5.6
5.4
—
—
5.9
7.9
4.5
8.3
15.3
5
5.4
5.8
—
5.2
12.6
—
—
4.5
8.6
15.7
5|
6.5
6.3
6.3
—
—
6.3
8.1
—
—
—
6
6.7
7.0
6.5
5.8
12.6
4.9
9.0
15.7
6£
7.0
7.2
—
—
—
—
—
—
7
7.4
7.4
7.0
6.1
12.6
5.4
9.4
16.2
71
8.1
7.4
7.9
—
—
—
—
—
8
—
—
8.1
6.7
12.6
5.4
9.4
16.2
8*
8.1
7.6
8.1
—
—
9
—
7.9
8.3
6.7
12.6
Qi
y2
8.5
8.5
8.5
—
—
10
9.0
9.0
8.8
7.6
14.0
10$
9.4
—
9.0
—
—
11
—
—
9.4
9.9
14.9
12
9.9
9.9
10.3
14.9
13
—
9.9
10.3
16.2
14
—
10.3
—
—
15
10.8
—
10.3
16.2
16
10.8
10.3
was found to be composed of two parts. The first can be described as the
shortening of the chromosomal fibers which moves the chromosomes towards the
poles. The second consists of the elongation of the spindle, resulting in a further
movement of the chromosomes.
In general, this picture of anaphase agrees with Belar's hypothesis which
174
HANS RIS
resolves anaphase into (1) the action of the "Zugfaser" and (2) that of the
"Stemmkorper." However, the chromosomal fibers, in the aphid at least, do not
attach to a continuous fiber ("Leitfaser"), but form direct connections from the
chromosome to the pole. No continuous fibers can be seen in this form. There
is also little in favor of a specific differentiation of the region between the daughter-
chromosomes into a "Stemmkorper." The intrusion of cytoplasmic granules into
the equatorial region of the spindle (page 168) is evidence that this part of the
TABLE II
Anaphase movement in primary spermatocytes of Tamalia (Ta), Protenor (P),
and Thelia (Th). d.k. = distance between kinetochores
Time
(minutes)
Ta 6a
23° C.
d.k.U)
Ta6b
23° C.
d.k.(M)
Ta 7
25°
d.k.(/0
P 2 25° C.
Th 1
25° C.
autosomes
d.k.(*0
X chromosome
d.k.(M)
0
4.5
4.5
4.5
3.6
2.9
4.9
1
4.9
5.4
4.9
3.8
—
—
2
5.4
6.3
—
4.0
2.9
5.4
1\
—
—
—
—
3.1
—
3
—
—
6.3
4.5
3.6
5.6
4
6.7
6.7
—
4.7
3.8
5.8
5
—
7.2
6.7
5.2
—
—
si
6
—
—
—
5.4
—
6.0
7
7.6
—
8.1
—
3.8
6.7
8
—
8.5
—
6.3
—
7.2
9
8.1
9.0
8.5
7.6
4.5
7.6
10
—
—
—
9.4
—
8.5
11
—
—
—
9.4
— -
9.0
12
8.5
9.4
—
9.4
—
9.0
13
—
—
9.4
10.8
4.7
—
14
—
—
—
11.2
5.4
9.4
15
9.9
. —
—
—
5.8
9.7
16
—
—
—
—
7.2
10.8
17
—
—
—
12.1
7.6
— .
18
—
—
—
—
7.9
10.8
19
10.8
. —
—
12.6
9.0
—
20
—
10.3
—
—
9.0
—
21
—
10.8
13.5
9.2
11.2
23
—
—
—
9.4
—
25
11.7
—
—
—
—
26
11.2
—
9.9
—
29
13.5
11.2
spindle is not a rigid "Stemmkorper," but rather less viscous than the rest of the
spindle. It is more likely that the spindle as a whole elongates, though probably
to a greater extent in the equatorial region. Only actual measurements can
clarify this point.
The shape of the chromosomes at anaphase indicates that the chromosomal
fibers exert a pull on the kinetochore. This is not only seen when the chromosomal
fibers shorten and bring the chromosomes to the poles, but also in the first
ANAPHASE MOVEMENT OF CHROMOSOMES
175
spermatocyte of Tamalia where spindle elongation alone moves the chromosomes.
The motion is therefore transmitted from the spindle to the chromosomes through
the chromosomal fibers. The elongating spindle then does not push the chromo-
somes apart, but separates the poles. The chromosomal fibers, which in some
way must be anchored to the polar regions then begin to pull at the spindle attach-
ments of the chromosomes (cf. Ris, 1942; Fig. 84-90).
In the aphid, Protenor, and Thelia the two components of the anaphase move-
ment are completely separated in time. How far can this type of movement be
generalized? Barber (1939) in staminal hair cells of Tradescantia found simple
S-shaped curves. He drew similar curves also through the points furnished
by Belar's photographs of anaphase in spermatocytes of the grasshopper
(Chorthippus). Belar's points are, however, so far apart that the lines drawn
through them are purely hypothetical; they may or may not be simple. In
TABLE III
Anaphase movement in spermatogonia of Protenor. d.k. = distance between kinetochores
Time
(minutes)
P4
25° C.
P5
25° C.
d.k.OO
Time
(minutes)
P 4
25° C.
d.k.U)
P 5
25° C.
d.k.GO
0
2.2
1.6
8
9.7
5.2
1
2
1
2.5
2.7
1.8
2.0
81
2
9
10.3
11.5
6.0
7.6
1|
2.9
2.0
Q—
11.5
8.1
2
3.1
2.2
io2
11.7
8.5
I '
3.4
3.6
2.2
11
11.7
9.0
9.0
42
3.8
4.9
2.2
11*
12
9.2
9.2
4§
5.6
—
13
10.3
5"
6.3
3.1
13?
11.7
5|
7.2
3.4
14
12.1
6
8.5
3.4
15
12.6
6*
7
9.0
9.2
3.6
4.0
16
18
13.0
13.0
71
9.4
4.9
Tradescantia staminal hair cells, as in other somatic plant cells, there is no elon-
gation of the spindle and cell (cf. Belar's photographs, 1929b). We may compare
therefore this entire anaphase movement with the first part of that in the aphid.
In both cases rather flat S-shaped curves are found. For the grasshopper pre-
liminary measurements have shown that the chromosome movement differs from
that of the aphid since the spindle begins to elongate before the shortening of the
chromosomal fibers is completed.
The anaphase curve with a distinct separation of the two components is found
in three Hemiptera and Homoptera, but in no other form analyzed so far. One
may therefore assume that it is related to the special kind of spindle apparatus
found in these forms, namely, the diffuse spindle attachment. Should this be
confirmed by further studies on other forms it would give additional evidence for
the functional importance of structures like chromosomal fibers still believed by
176
HANS RIS
some investigators to be artifacts. It would also be an interesting example of how
variations in cellular processes are related to differences in structure.
The behavior of the X chromosome in the first spermatocyte of Protenor is of
great interest. Chromosomal fibers are present in metaphase and anaphase, but,
as the analysis of the movement in a living cell shows, they are hindered in their
normal functioning so that the X chromosome lags behind the autosomes on its
way to the poles. This provides a mechanism for individual movements of
chromosomes. A similar condition may be responsible for the lagging of specific
chromosomes in elimination divisions of Sciara, Oligarces, etc.
The velocity of the chromosomes at anaphase is of great interest. The maxi-
mum velocities in the various divisions studied are brought together in table IV.
The velocities due to the shortening of the chromosomal fibers and spindle
elongation are recorded separately. The greatest velocity in embryonic cells of
TABLE IV
Maximum velocities of chromosomes. Micra/minutes. 23-26° C.
Somatic mitosis
Spermatogonia
Meiosis I
Meiosis II
chromosomal
fibers
spindle
chromosomal
fibers
spindle
chromosomal
fibers
spindle
chromosomal
fibers
spindle
Tamalia
0.7-2
0.3-1.1
—
0.3
0.9-1.2
0.4-1.1
Protenor
1.3-1.6
0.3-0.5
0.9
0.7
Thelia
0.4
0.5
Tradescantia
(Barber 1939)
1.2
(20° C.)
Tamalia is 2 ju per minute, or about 3 mm. in 24 hrs. As comparison the maxi-
mum velocity in Tradescantia staminal hair cells reported by Barber (1939) is
added to the table.
CONCLUSIONS
The character of the chromosome movement at anaphase varies in different
groups of organisms. It is possible to describe these differences as modifications
in the behavior of components of the mitotic apparatus, such as chromosomal
fibers and spindle body. Thus in Tradescantia staminal hair cells there is only
the movement to the poles, in the first meiotic division of Tamalia only the
elongation of the spindle (diagram Fig. 9). In regular divisions of Hemiptera
and Homoptera the action of chromosomal fibers and spindle elongation are
separated in time (diagram Fig. 8), in the grasshopper, however, they act simul-
taneously. These functional differences are correlated with variations in the
spindle structure (diffuse against localized spindle attachment).
Measurements of chromosome movement such as those reported by Barber
(1939) and in this paper represent a first step in the analysis of anaphase, namely
ANAPHASE MOVEMENT OF CHROMOSOMES
177
a quantitative description of the processes observed in the cell. The movement
must then be separated into its components and related to the cellular structures
which are found to be essential for regular separation of chromosomes (kinetochore,
chromosomal fibers, spindle, etc.). A theory of chromosome movement must be
tf)
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FIG. 8
FIG. 9
TIME
TAMALIA EMBRYONIC CELLS TAMALIA PRIMARY SPERMATO-
TAMALIA SECONDARY SPER— CYTES
MATOCYTES
PROTENOR SPERMATOGONIA
PROTENOR PRIMARY SPER —
MATOCYTES
THELIA PRIMARY SPERMATO-
CYTES
FIGURES 8 and 9. Diagrams illustrating the chromosome movement in forms with diffuse
spindle attachment. 8: the typical anaphase curve. 9: the exceptional curve in primary sperma-
tocytes of Tamalia.
established first on a biological plane, accounting for the many modifications of
anaphase as variations of these mitotic organelles. Finally an experimental
analysis of the nature of these structures and the changes they undergo during
mitosis can provide an empirical basis for a physico-chemical theory of mitotic
movement.
178 HANS RIS
SUMMARY
1. The movement of chromosomes at anaphase was measured in living cells of
Tamalia, Protenor and Thelia. The distance between the separating chromosomes
plotted against time produces curves which describe accurately the chromosome
movement. In embryonic cells and secondary spermatocytes of Tamalia,
spermatogonia and primary spermatocytes of Protenor, and a primary spermato-
cyte of Thelia the curves consist of two S-shaped components separated by a
plateau. The second part of the movement coincides with the elongation of the
cell.
2. In stained sections the length of chromosomal fibers and the spindle was
measured at various stages of chromosome separation. A comparison with the
data from living cells shows that in the first part of anaphase the chromosomal
fibers shorten, i.e., the chromosomes approach the poles. In the second part the
spindle elongates and thus produces a further movement of the chromosomes.
3. The chromosome movement in the otherwise exceptional anaphase of pri-
mary spermatocytes in Tamalia is characterized by a simple unbroken curve.
Measurements on'stained cells demonstrate that the movement is due entirely to
spindle elongation. The chromosomal fibers remain constant in length and the
chromosomes therefore do not approach the poles.
4. Since the double curve was found in all Hemiptera and Homoptera studied
but not in the grasshopper (unpublished results) this type of anaphase movement
is probably related to the diffuse spindle attachment found in these insects. This
points out the functional significance of structural variations.
5. The curves for the primary spermatocyte of Protenor show that the lagging
of the daughter chromosomes of the univalent X chromosome is due to an ab-
normal first part of the movement. This indicates some impairment in the
functioning of their chromosomal fibers. The exceptional behavior of a chromo-
some can thus be traced to one particular factor of the anaphase movement.
LITERATURE CITED
BARBER, H. N., 1939. The rate of movement of chromosomes on the spindle. Chromosoma, 1:
33-50.
BELAR, K., 1929a. Beitrage zur Kausalanalyse der Mitose. II. Untersuchungen an den Sperma-
tocyten von Chorthippus (Stenobothrus) lineatus Panz. Roux' Arch. f. Entw. mech.,
118: 359-484.
BELAR, K., 1929b. Beitrage zur Kausalanalyse der Mitose. III. Untersuchungen an den Staub-
fadenhaarzellen und Blattmeristemzellen von Tradescantia virginica. Z. Zellforsch.,
10: 73-134.
HuGHES-ScHRADER, S., AND H. RIS, 1941. The diffuse spindle attachment of coccids, verified by
the mitotic behavior of induced chromosome fragments. Jour. Exp. Zool., 87: 429-456.
RIS, H., 1942. A cytological and experimental analysis of the meiotic behavior of the univalent
X chromosome in the bearberry aphid Tamalia (= Phyllaphis) coweni (Ckll.). Jour.
Exp. Zool., 90: 267-330.
SCHRADER, F., 1935. Notes on the mitotic behavior of long chromosomes. Cytologia, 6: 422-430.
Vol. 85, No. 3 December, 1943
THE
BIOLOGICAL BULLETIN ^
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE OSMOTIC PROPERTIES OF CYTOPLASM 1C GRANULES
OF THE SEA URCHIN EGG1
DANIEL L. HARRIS
(Zoological Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania, and
The Marine Biological Laboratory, Woods Hole, Massachusetts)
INTRODUCTION
In main- cells, a large part of the cytoplasm consists of numerous granules
of various types. Undoubtedly they have a real functional significance, but
little is yet known of the roles which they may play in cellular processes. This
lack of knowledge may be due to the fact that, ordinarily, granules are rather
inaccessible to experimental treatment. It was therefore thought worth while
to attempt to isolate the granules and study them outside the living cell. This
method has the advantage that it enables one to add reagents in known concen-
trations, and to be certain that they are affecting the granules directly and not
secondarily through effects upon the cell itself.
Although most granules appear as solid particles, many may actually be
minute vacuoles. That this is true of the pigment granules of the Arbacia egg
was concluded by Chambers (1935) who found that the pigment escapes when
these granules are punctured with a micro-needle. In preliminary studies, I
was able to confirm this observation and to provide additional evidence that
the pigment granules are actually vacuoles. The most cogent part of this evi-
dence is the fact that in the presence of calcium, magnesium, and strontium ions,
the pigment granules (and some colorless granules) coalesce with each other to
form large fluid vacuoles from which the pigment soon diffuses, revealing small
particles inside in active brownian movement. It is difficult to understand how
two particles could coalesce in this manner unless they were vacuoles initially.
Photomicrographs of this interesting reaction in isotonic CaCl2 are shown in
Figure 1.
Such minute granules or vacuoles should exhibit osmotic activity. That
they do was concluded by Lewis and Lewis (1915) who actually observed swelling
and shrinking of mitochondria in tissue culture cells placed in anisotonic media,
and by Costello (1939) who found that the formed components of the Arbacia
egg occupy 41 per cent of the total volume of the egg, while the osmotically
1 Presented to the Faculty of the Graduate School of the University of Pennsylvania in
partial fulfillment of the requirements for the Degree of Doctor of Philosophy.
179
180
DANIEL L. HARRIS
.
.S O
•
O
ba
= "= -B
'x rt -C
O jj C
«>
O
c
a a
0) OJ
_2 —
~ e
o •-
as -
c
O ••
V4-r O
CYTOPLASMIC GRANULES OF SEA URCHIN EGG 181
inactive volume is only 7-14 per cent according to McCutcheon, Lucke and
Hartline (1935).
The present paper presents direct evidence that the pigment vacuoles of the
Arbacia egg are osmometers. They do not, therefore, constitute part of the
osmotic dead space postulated by McCutcheon, Lucke and Hartline (1935).
Agreement with the Boyle- Van't Hoff law is rather good but certain discrepancies
point to the conclusion that osmotically active material, presumably salt, leaks
out during the course of the swelling. Some data is given for other types of
granular inclusions.
It is a pleasure to express my appreciation to Doctor L. V. Heilbrunn for his
encouragement and stimulating advice.
PREPARATION OF MATERIAL
Suspensions of granules or vacuoles in vitro may readily be obtained from sea
urchin eggs if certain precautions are taken. The solutions used must be neutral
or acid, isotonic, and free from calcium. In alkaline solutions or in hypotonic
solutions the vacuoles undergo lysis. In solutions containing calcium (or
magnesium or strontium in high concentration) the protoplasm escaping from a
ruptured cell clots. This reaction, called by Heilbrunn (1928) the surface
precipitation reaction, must be avoided, inasmuch as many vacuoles are trapped
in the clotted protoplasm and others lyse or coalesce with each other. The
necessary precautions may be conveniently taken by using an isotonic solution
of sodium citrate (0.35 M). This solution has several additional advantages.
It does not induce cytolysis and eggs washed in it become very fragile and easily
ruptured.
Eggs were collected from 10-50 sea urchins by allowing them to shed into
sea water. The shedding reaction was hastened by the addition of isotonic
KC1 to the exposed ovaries according to the method of Palmer (1937). The
eggs were concentrated by centrifuging and washed with 0.35 M sodium citrate.
After two washings, most of the eggs become very fragile and may be broken
readily by squirting them in and out of a pipette. The vacuoles themselves are
rather sensitive, and it was found impossible to rupture the most resistant eggs
(about 25 per cent) without simultaneously destroying many of the vacuoles.
The crude suspension of all types of granules or vacuoles resulting from this
treatment may be used for many experiments. However, if desired, the various
components may be separated out by differential centrifuging. A wide range of
conditions may be used to accomplish this; but, on the whole, it is somewhat
better to use low centrifugal forces for a long time rather than high forces for a
short time, since if the vacuoles become tightly packed in the bottom of the
centrifuge tube it is difficult to re-suspend them without causing serious breakage.
The following scheme has proved satisfactory. Unbroken cells are removed
rapidly by filtering through coarse filter paper under light suction. The whole
brei is then centrifuged with an International Centrifuge, size 1 type SB, or
size 2. The pigment vacuoles are thrown down in about 30 minutes at 1000 rpm
(189 X gravity, g). Yolk sediments at 2000 rpm (755 g) in one hour. At
3000 rpm (1698 g) very small particles are thrown down in considerable quantity
in three to five hours. Fat granules rise to the top and are readily removed.
182
DANIEL L. HARRIS
If centrifuging is prolonged or if higher forces are available, it is possible to
obtain granule-free cytoplasm. In each fraction there is a certain amount of
contamination. This may, for the most part, be removed by re-suspending the
particles in fresh citrate and repeating the original centrifuging. After separa-
tion, the granules or vacuoles are washed with fresh isotonic citrate to remove
traces of non-granular protoplasm.
.m
.110 .228 .W .263 .760 .290 .3/5
MOLARITY OF SODIUM CITRATE
.33*
.yso
FIGURE 2. Lysis of pigment vacuoles in hypotonic sodium citrate. Circles represent data
obtained by counting vacuoles microscopically; triangles, data obtained by colorimetric method
explained in text. Curve is a cumulative projection of a normal or probability curve.
Another method of separating the constituents was sometimes employed.
Concentrated solutions of sodium citrate may be prepared with a specific gravity
higher than that of any of the granules, or with a specific gravity intermediate
between that of any two types. By properly choosing the concentration,
separation may be readily accomplished by placing a layer of concentrated
citrate beneath the suspension of mixed particles before centrifuging. The
proper concentration must be determined with each preparation because of
variability in the specific gravity of the particles. Unfortunately, the pigment
CYTOPLASMIC GRANULES OF SEA URCHIN EGG
183
vacuoles undergo lysis with this treatment and, therefore, cannot be recovered
intact. The yolk and the small granules do not seem to be adversely affected.
RESULTS
In hypotonic solutions pigment vacuoles undergo lysis. Microscopic observa-
tion of the reaction shows little detail, but it is possible to see that, after a short
time, the pigment suddenly leaks out of the vacuole which then fades from view,
leaving an indistinct ghost. There is no obvious rent in the vacuolar membrane,
nor does the pigment stream out from a localized spot. On the contrary, it
appears to diffuse through the entire membrane much as hemoglobin diffuses
out of blood cells in hemolysis. Indeed, the phenomena of vacuolar lysis and
hemolysis seem to be rather comparable, and the methods which have been
used to study hemolysis may be applied here.
TABLE I
Times of lysis (seconds) of pigment vacuoles
Molarity Na3 citrate
0.17.SO
0.1025
0.2100
0.2275
0.2450
0.2625
Osmotic pressure
(Atmospheres)
10.50
11.49
12.47
13.46
14.44
15.43
Per cent
lysis
I.ytic osmotic
pressure
5
16.42
1.48
1.76
1.84
2.30
2.63
5.66
10
15.63 1.65
1.91 2.45
2.81
4.68
15.40
15
15.43 1.71
2.16 2.72
3.98
7.93
20
15.04 1.80
2.88
3.72
4.85
14.50
30
14.64 2.56
2.96
3.89
5.38
23.40
35
14.44 3.54 5.15 6.51
10.30
40
14.26
3.50
5.43
7.35
12.50
50
13.95 6.83
7.80
12.70
28.70
65
13.46
18.80
29.90
40.60
1
If the vacuoles are osmometers, there should be more lysis in a very dilute
solution than in a mildly dilute solution. This was studied in the following
manner. Aliquots of the crude suspension were added to various concentrations
of sodium citrate. Samples taken to determine the percentage of lysis were
placed in a chamber of definite volume and uniform depth (Leitz dark field
chamber) and the pigment vacuoles in a given area were counted. A 4 mm.
objective and 10X ocular gave adequate definition. The results of these counts
are represented by the circles in Figure 2. It will be noted that lysis increases
as the external solution is made more dilute. Lysis is practically complete in
0.175 AI sodium citrate. The curve is a cumulative projection of a probability
curve, and the fit is close enough to indicate that the vacuoles are "normally'
distributed in their resistance to lysis. The same type of curve is found in
osmotic hemolysis, and is interpreted in the same wray.
A quicker and easier method of estimating the amount of lysis is afforded by a
colorimetric method. This depends upon certain properties of the pigment,
echinochrome, contained within the vacuole. According to Kuhn and Wallenfels
DANIEL L. HARRIS
^pl********11*
FIGURE 3.
CYTOPLASMIC GRANULES OF SEA URCHIN EGG
185
(1939), this pigment is a polyhydroxyquinone bound to a protein. In the acid
form it is red in color, and this is presumably the condition in which it exists
within the vacuoles. The salts are variously colored. In sodium citrate at
pH 7.4 the pigment escaping from the vacuoles turns first to a dirty brown and
too
.000
.035
.070
JOS .I'M .175 .2/0 .2*5
MOLARITY OF SODIUM CITRATE
.200
.375
FIGURE 4. Lysis of yolk granules in hypotonic sodium citrate. Circles represent data
obtained with the photoelectric method. Triangles show the increment of lysis with dilution.
The curves are empirical.
ultimately to a clear green. Intermediate colors are obtained which depend
upon the amount of lysis. Standards may be prepared by making mixtures of
suspensions of intact vacuoles with suspensions of vacuoles lysed with hypotonic
citrate and brought back to isotonicity with concentrated citrate. The per-
FIGURE 3. Photographic records of granule lysis with Parpart's photoelectric method.
The addition of the granules to the hypotonic solution is marked by a sudden drop in light trans-
mission, here a thin vertical line (retouched). As lysis proceeds there is a rapid increase in light
transmission followed by a slower increase to equilibrium. White vertical lines mark second
intervals. Upper row: lysis of yolk granules in 0.280 M, 0.140 M, 0.035 M sodium citrate.
Lower row: lysis of small granules (mitochondria?) in 0.280 M, 0.175 M, 0.105 M sodium citrate.
186
DANIEL L. HARRIS
centage lysis in any experimental suspension may be determined by comparing
the color with that of the standards. This was done at the same time the counts
were made, and the results are shown by the triangles in Figure 2. Agreement
between the two methods is good and the colorimetric method was used thereafter.
With the colorimetric method it is possible to study the kinetics of the reaction.
The time taken to achieve a certain degree of lysis in a given hypotonic solution
can be measured by determining the time required to reach a certain color.
This was done with a stop-watch and visual inspection. The results are given
in Table I. In this table the times required to attain different percentages of
lysis in various concentrations of hypotonic sodium citrate are recorded. It is
apparent that in the more dilute solutions, not only is the degree of lysis greater
but the speed of the reaction is very much greater as well. In a more dilute
solution the osmotic gradient is greater; water will therefore enter more rapidly;
and the vacuoles will swell to the lytic size in a shorter time.
TABLE II
Times of lysis (seconds) of yolk granules
Molarity Na3 citrate
0.00
0.035
0.070
0.105
0.140
0.175
0.210
0.245
0.280
Osmotic pressure
(Atmospheres)
0.00
2.61
4.58
6.56
8.53
10.50
12.47
14.44
16.42
Per
cent
lysis
Lytic
osmotic
pressure
27
18.38
0.34
0.41
0.46
0.41
0.62
0.76
1.02
1.46
6.50
51
16.42
0.44
0.56
0.68
0.75
1.07
1.42
2.07
5.90
68
14.44
0.55
0.86
1.25
2.12
4.90
71
12.47
0.58
1.05
1.91
4.80
73
10.50
0.61
1.29
3.80
78
8.53
0.66
2.68
81
6.56
0.79
14.18
86
4.58
0.87
92
2.61
2.18
A few observations were made of the lysis of purified yolk granules using
the photoelectric method of Parpart (1935).2 Light from a constant source is
sent through a chamber and is measured by means of a Photronic cell and a
Kipp-Mall galvanometer with photographic recording. Typical records are
shown in the top row of Figure 3. On the addition of 50 mm.3 of granule sus-
pension to the dilute salt solution in the chamber there is a rapid drop in the
light transmission. As lysis proceeds, more light passes through the suspension
and the galvanometer tends to return to its original position. The results of
these experiments are summarized in Figure 4 and Table II.
Similar records were made with the smallest granules (mitochondria?).
Unfortunately, these particles tend to clump together and it is exceedingly
difficult to obtain uniform samples. Analysis of the data is at present impossible.
Typical records are, however, shown in the bottom row of Figure 3.
2 I am very grateful to Doctor A. K. Parpart for the loan of his own apparatus.
CYTOPLASMIC GRANULES OF SEA URCHIN EGG
187
DISCUSSION
The pigment vacuole:
While the above data indicate clearly that the pigment vacuoles are os-
mometers, a closer analysis is desirable. We would like to know if the Boyle-
Van't Hoff law is obeyed. Equations suitable for testing this have been derived
by Jacobs (1932) for the comparable case of the osmotic hemolysis of blood.
For the general case :
KAt _ po pop - - pP po / \_ 1
To" ~ p*np0p-- p0P ' P\p0" p
and for the special case where cells (or vacuoles) swell in distilled water:
KAt __ po
~\7 1 ^~>
l/o 2 \/>-
A. •>
/Jo-
in these equations, K is the permeability constant, a measure of the volume
of water entering the vacuole through a unit area in a unit time under a unit
osmotic gradient. A is the surface area, Vo the initial volume of the vacuoles.
TABLE III
Permeability of the pigment vacuoles to water (A"')
Molarity Naa citrate
0.1750
0.1925
0.2100
0.2275
0.2450
0.2625
Osmotic pressure
(Atmospheres)
10.50
11.49
12.47
13.46
14.44
15.43
Per cent
lysis
Lytic osmotic
pressure
5
16.42
.022
.021
.024
.024
.027
.019
10
15.63
.027
.027
.026
.029
.024
.016
15
15.43
.026
.026
.025
.022
.016
20
15.04
.031
.023
.025
.023
.012
30
14.64
.025
.026
.025
.025
.012
35
14.44
.020
.016
.017
.010
40
14.26
.021
.017
.016
.014
50
13.95
.012
.013
.011
.008
65
13.46
.005
.004
.004
P is the osmotic pressure of the external solution, an experimentally controlled
variable. The volume of the external solution is very large in comparison to
the total volume of the vacuoles, so that the external osmotic pressure does not
change during the course of an experiment. Now, p0 is the osmotic pressure of
the solution inside the vacuole initially, p the osmotic pressure inside the vacuole
at time /. The osmotic pressure within the vacuole may be assumed to be that
of the solution with which it is in equilibrium. Initially, this is equivalent to
0.35 M sodium citrate, or 20.36 atmospheres (calculated from data of Hitchcock
and Dougan, 1935). We are interested only in a particular value of p, that is
PL corresponding to IL- This is the osmotic pressure inside the vacuoles at the
moment of lysis, and is presumed to be exactly equivalent to the osmotic pressure
188
DANIEL L. HARRIS
of the solution which causes that degree of lysis, i.e., the solution with which
it is in equilibrium.
For the purpose of determining agreement with the Boyle-Van't Hoff law,
it is not necessary to know the exact volume, V0, or surface area, A, since these
are constant and may be combined with K, the true permeability constant, to
give a new constant K'. If the same value of K' is found for all concentrations,
it may be concluded that the vacuoles obey the Boyle-Van't Hoff law.
The data necessary for this calculation may be obtained from Figure 2 and
Table I and the results are given in Table III for several concentrations and a
number of degrees of lysis.
It will be noted that when 5 to 30 per cent lysis occurs, all the values of K'
lie around 0.025 except for the last figure in each row, which is close to equilibrium
.175 ./93 .2/0 .228 .2*5 .263
MOLARITY OF SODIUM CITRATE
.260
FIGURE 5. Lylic time of pigment vacuoles in hypotonic sodium citrate. Circles represent
5 per cent lysis, triangles 15 per cent lysis, and squares 35 per cent lysis. Curves are based on
equation given in the text assuming K' = 0.025.
and may be disregarded. From 35 to 65 per cent lysis, K' falls to very low
levels. It may be further noted that in many of the rows there is a gradual
decrease of K'. This is not especially serious except in more advanced degrees
of lysis.
From these data it seems justifiable to conclude that up to 30 per cent lysis,
K' is essentially constant and the pigment vacuoles therefore obey the Boyle-
Van't Hoff law. This can be shown in a somewhat more convincing manner by
a comparison of the observed rate of lysis with the theoretical rate, assuming a
value of K' = 0.025. Figure 5 shows the theoretical curves for three different
CYTOPLASMIC GRANULES OF SEA URCHIN EGG 189
degrees of lysis. It will be noted that for 35 per cent lysis there is no agreement
between the curve and the plotted data, although there would have been had
another value of K' been chosen. Agreement of the data with the other two
curves is good over a limited range, although large discrepancies exist at high
concentrations of sodium citrate.
The reasons for the discrepancies which are found may now be considered.
Either the permeability constant falls during the course of swelling or else the
rate of lysis is very much slower for high degrees of lysis than is expected theo-
retically. With the data at hand, it is impossible to prove either of these possi-
bilities, but the latter seems far more probable. The simplest interpretation is
that osmotically active materials, presumably salts, leak out from the vacuoles,
thus reducing the osmotic gradient and causing water to enter more slowly.
This leakage of osmotically active materials does not include the echinochrome-
protein complex. Obviously, within the cell the pigment does not normally
diffuse out from the vacuole, or the vacuoles would contain no pigment. In
vitro, the strict parallelism between the number of vacuoles lysed and the amount
of pigment released as determined colorimetrically shows that there is no escape
of echinochronie from the vacuoles until lysis occurs.
In deriving the equations, Jacobs made certain assumptions which have
likewise been made here. It is instructive to consider these assumptions, particu-
larly with the view of determining if any of them may aid in explaining the
discrepancies. It is first assumed that the surface area of the vacuole is not
changed during the course of swelling. It is clear that in the original permeability
equation, dV/dt = KA(p -• P), the rate of swelling is directly proportional to
surface area. Thus if there is any significant increase in surface area during
swelling, the vacuoles ought to lyse more rapidly than expected after the initial
stages. Actually, the rate is slower. This does not mean that increase in
surface area does not occur; but if such increase does occur, it will not serve to
explain the divergent results and it is therefore a refinement which can be
neglected.
The assumption is also made that the initial volumes of the individual vacuoles
are the same. This is by no means true since they vary in size from one to two
micra in diameter. This variation should not affect the results unless there is a
correlation between the initial size and the lytic concentration. Such a condition
would be true if it were assumed that the vacuoles undergo lysis only when they
swell to a lytic volume constant for all vacuoles. In that case the largest should
undergo lysis first. Progressively smaller granules would be destroyed in the
course of time in very dilute solutions. Remembering that K' — KA/V0 it is
apparent that if the diameter, which is a measure of both VQ and A, is assumed
larger than it actually is, K' will appear fallaciously low. This would be the
situation for high degrees of lysis. A correction for varying initial volumes
would, therefore, reduce some of the discrepancies. It would not, however,
raise to the extent necessary the low values of K' found with high degrees of
lysis, nor would it affect at all the drift of K' in high concentrations.
In the absence of concrete data, this possibility of explaining the discrepant
results cannot be disproved. However, it seems unlikely that there is a strict
correlation between initial volume and lytic concentration in view of the fact
that the same type of curve as is shown in Figure 1 is obtained in hemolysis.
190 DANIEL L. HARRIS
Erythrocytes are quite uniform in size. Furthermore, a simple calculation will
show that no strict relation, such as Boyle's law, is likely to exist between initial
volume and lytic concentration. If vacuoles two micra in diameter are assumed
to break in 0.280 M sodium citrate, it may be calculated by Boyle's law that
vacuoles one micron in diameter ought to lyse in 0.035 M. Actually, lysis is
complete in 0.175 M.
It seems more reasonable to assume that the vacuoles have to swell a certain
proportion of their original volume before lysis occurs than that the lytic volume
is constant for all size vacuoles. On this assumption, the lytic concentration for
all of the vacuoles would be the same. In view of these reasons for considering
initial volume and lytic concentration independent, there seems no necessity for
assuming that a correlation exists. In that event, a variation in the initial
volume will have no effect upon K', and V0 may legitimately be considered
constant.
It is of some interest to calculate the true permeability constant. This may
be very simply done if we remember that KA/Vo = K' . Since the pigment
vacuoles are spherical, both VQ and A can be expressed in terms of the diameter,
D, and multiplying by 60 to change seconds into minutes we obtain:
=
Taking 0.025 as near the true value of Kr, and D — 1 • - 2p, K falls in the range
of 0.25-0.50 with an average value of 0.38 cubic micra of water entering the
vacuole per square micron of surface area per minute per atmosphere difference
in osmotic pressure.
These values of the permeability constant should not be taken too seriously.
They are of interest only in indicating the order of magnitude. The initial
osmotically active volume is not exactly known. Moreover, it should be noted
that these measurements were made in vitro and in the presence of the citrate
ion. Both of these conditions might well influence the permeability. Never-
theless, the permeability constant is probably somewhat higher than that of the
cell as a whole. Lucke, Hartline and McCutcheon find values of 0.087 for
endosmosis and 0.141 for exosmosis. The pigment vacuoles will tend toward
osmotic equilibrium with the rest of the cell and cannot constitute part of the
osmotic dead space postulated by McCutcheon, Lucke and Hartline to explain
divergencies of the osmotic behavior of the Arbacia egg. This dead space
probably consists of fat granules, dissolved proteins, or the membranes, etc.,
which surround the cell and the various formed components in it.
If the interpretation that the vacuoles are leaky is correct, they should be in
equilibrium with the rest of the cell in regard to salts and organic substances,
e.g., metabolites. The pigment vacuoles undergo lysis in solutions of urea,
acetamide, sucrose, ethylene glycol, etc., indicating they are permeable to these
substances. Whether these or other substances added to the cell would penetrate
the cell and all its constituents at the same rate, cannot be answered until com-
parative figures of the permeability constants of these substances are available.
This information may ultimately have important bearing on problems of cell
metabolism. Further experiments are planned.
CYTOPLASMIC GRANULES OF SEA URCHIN EGG
191
Yolk granules:
The interpretation of the behavior of the yolk granules in hypotonic solutions
is difficult. An inspection of the curve given in Figure 3 indicates that the
reaction proceeds in two stages. This is especially clear from the curve marked
with the triangles which is the increase in lysis with each increment of dilution.
It is possible that down to about 0.245 M, the yolk particles are lysed osmotically,
but that in lower concentrations some other process is operating.
TABLE IV
Permeability of yolk granules to water (A"')
Molarity Naa citrate
0.00
0.035
0.070
0.105
0.140
0.175
0.210
0.245
0.280
Osmotic pressure
(Atmospheres)
0.00
2.61
4.58
6.56
8.53
10.50
12.47
14.44
16.42
Per
cent
lysis
Lytic
osmotic
pressure
27
18.38
.0173
.0160
.0161
.0210
.0169
.0164
.0158
.0156
.0060
51
16.42
.0321
.0281
.026§
.0285
.0244
.0230
.0215
.0122
68
14.44
.0475
.0346
.0273
.0196
.0106
71
12.47
.0769
.0491
.0322
.0159
73
10.50
.1234
.0691
.0289
78
8.53
.1996
.0605
81
6.56
.3211
.0220
86
4.58
.6921
92
2.61
1.1394
The rate of the reaction increases with dilution, as shown in Table II. This
is consistent with the osmotic hypothesis. However the results of calculating
K' (Table IV) do not lend support to this hypothesis. K' does not have the
same value for all concentrations and all degrees of lysis. Indeed, it ranges
from 0.0060 to 1.1394 in a very regular manner. Seemingly a simple osmotic
hypothesis cannot explain these results. Perhaps an actual solution of the yolk
particles in hypotonic solution occurs.
SUMMARY AND CONCLUSIONS
A method has been developed with which it is possible to obtain relatively
pure suspensions of cytoplasmic granules in good physiological condition. With
these preparations, some of the properties of the granules have been studied.
It seems clearly established that the pigment granules are actually vacuoles,
and that they show osmotic activity. No certain conclusions can be drawn
about the yolk granules.
During the first few seconds of swelling, the pigment vacuoles show rather
good agreement with the Boyle-Van't Hoff law. However, as exposure to
hypotonic solutions continues, the rate of lysis falls below the expected value.
This is interpreted as due to a leakage of osmotically active materials, probably
salts, from the vacuoles, thus reducing the osmotic gradient, and therefore
192 DANIEL L. HARRIS
causing water to enter more slowly. The permeability constant of the pigment
vacuoles seems to be somewhat higher than that of the cell as a whole. The
vacuoles therefore tend toward osmotic equilibrium with the rest of the cell at
all times. They do not constitute part of the osmotic dead space found by
McCutcheon, Lucke and Hartline in the Arbacia egg.
If the interpretation that the granules are leaky is correct, they will tend
toward equilibrium with the rest of the cell in respect to salts and to organic
substances, although they are not permeable to the echinochrome-protein
complex.
This information may eventually be of considerable importance for our
understanding of the intimate problems of cellular metabolism and activity.
Ultimately, students of cellular permeability, metabolism, or many of the other
problems of cell physiology will have to consider the individual properties of all
the components of the cell.
LITERATURE CITED
CHAMBERS, R., 1935. The living cell. Chap. I, Textbook of Biochemistry, Harrow and Sherwin,
Philadelphia.
COSTELLO, D. P., 1939. The volumes occupied by the formed cytoplasmic components in marine
eggs. Physiol Zool., 12: 13-20.
HEILBRUNN, L. V., 1928. The colloid chemistry of protoplasm. Berlin.
HITCHCOCK, D. I., AND R. B. DOUGAN, 1935. Freezing points of anti-coagulant salt solutions.
/. Gen. Physiol., 18: 485-490.
JACOBS, M. H., 1932. Osmotic properties of the erythrocyte. III. The applicability of osmotic
laws to the rate of hemolysis in hypotonic solutions of non-electrolytes. Biol. Bull.,
62: 178-194.
KUHN, R., AND K. WALLENFELS, 1939. Uber die chemische Natur des Stoffes, den die Eier
des Seeigels (Arbacia pustulosa) absondern, um die Spermatozoen anzulocken. Ber.
des. deutsch. chem. Ges., 72: 1409.
LEWIS, M. R., AND W. H. LEWIS, 1915. Mitochondria and other cytoplasmic structures in
tissue culture. Am. J. Anal. 17: 339. |
LUCRE B., H. K. HARTLINE, AND M. MCCUTCHEON, 1931. Further studies on the kinetics of
osmosis in living cells. /. Gen. Physiol., 14: 405-419.
MCCUTCHEON, M., B. LUCKE, AND H. K. HARTLINE, 1931. The osmotic properties of living
cells (eggs of Arbacia punctulata). /. Gen. Physiol., 14: 393-404.
PALMER, L., 1937. The shedding reaction in Arbacia punctulata. Physiol. Zool., 10: 352-367.
PARPART, A. K., 1935. The permeability of the mammalian erythrocyte to deuterium oxide
(heavy water). J. Cell. Comp. Physiol., 7: 153.
THE RADIOSENSITIVITY OF EGGS OF ARBACIA PUNCTULATA IN
VARIOUS SALT SOLUTIONS l
KARL M. WILBUR2'3 AND RICHARD O. RECKNAGEL
(The Marine Biological Laboratory, Woods Hole; Department of Zoology and Entomology, The Ohio
State University; and the Zoological Laboratory, University of Pennsylvania)
A variety of experimental procedures has been shown to alter the sensitivity
of cells to x-rays and radium. Resistance to radiation can be increased by a
reduction of oxygen (Crabtree and Cramer, 1933; Mottram, 1935; Anderson and
Turkowitz, 1941); by the use of appropriate concentrations of ammonia (Zirkle,
1936; Marshak, 1938); CO2 and H2S (Zirkle, 1936, 1940, 1941) and by addition of
protein to the medium in which the cells are immersed (Evans et al., 1941). Con-
versely, certain agents increase the radiosensitivity of biological material (see
Scott, 1937). The present study has been carried out to ascertain whether
alteration of the salt environment, which will in turn cause changes in the ionic
composition, and to some extent the colloidal state of the protoplasm, will
influence the action of x-radiation on the living cell.
Three solutions have been used to alter the ionic composition of the egg:
isotonic potassium citrate; a mixture of isotonic MgCU and sea water; and a
mixture of isotonic CaCh and sea water. Potassium citrate is of particular
interest in this connection in that it will remove a large part of the calcium from
the cell and at the same time is relatively non-toxic. A further point of interest
lies in its inhibition of the reactions initiated by ultra-violet light in the Nereis
egg (Heilbrunn and Wilbur, 1937). Magnesium, like citrate, is inhibitory with
respect to ultra-violet action (Wilbur, 1939). Calcium is antagonistic to both
citrate and magnesium in many reactions of living material and so has been
studied along with these two ions in the present work.
METHODS
Prior to irradiation 0.1 to 0.2 cc. of concentrated eggs was added to 40 cc.
of the experimental solution or sea water for various periods. The eggs were
then transferred to small plastic dishes for irradiation. Following irradiation
0.15 to 0.25 cc. of solution containing the irradiated eggs was placed in 250 cc.
of sea water to remove the experimental solution; and approximately 6 minutes
later the eggs were transferred to a second dish of sea water which contained
sperm. The time required for 50 per cent of the eggs to complete first cleavage
was determined by fixing samples at 2-minute intervals in 1 per cent or 2.5 per
cent formaldehyde in sea water after examination of the eggs showed that cleavage
had begun. In a few instances in which the cleavage time occurred very slowly
1 A grant from the Graduate School of The Ohio State University is gratefully acknowledged.
2 A portion of this work was carried out during the tenure of a Rockefeller Fellowship.
3 Present address: Physiology Dept., Dalhousie University, Halifax, Canada.
193
194
K. M. WILBUR AND R. O. RECKNAGEL
samples were fixed at 3-minute intervals.4 By this method one can estimate
the time to 50 per cent cleavage in normal eggs within one or two minutes.
After very large doses of x-rays many of the eggs show multipolar cleavage, and
it is not always easy to decide the exact time at which the cleavage furrows have
cut completely through the egg. In such cases determinations of the time of
cleavage are accordingly somewhat less accurate. In most experiments the
TABLE I
Effect of x-radiation on Arbacia eggs following treatment with 0.35 M potassium citrate
Cleavage time of non-irradiated
Cleavage time of eggs receiving
Cleavage time of eggs receiving
eggs
30,400 r
53,200 r
Exp.
No.
Eggs in
sea water
Eggs treated with
potassium citrate
Eggs in
sea water
Eggs treated with
potassium citrate
Eggs in
sea water
Eggs treated with
potassium citrate
through-
out
For 30 min.
For 60 min.
through-
out
For 30 min.
For 60 min.
through-
out
For 30 min.
For 60 min.
I
II
Ill
IV
V
VI
VII
VIII
IX
X
1.
45
43
207
166
2.
41
42
118
110
3.
41
43
148
130
4.
42
42
175
150
5.
44
43
176.5
152
6.
44
42
159
147
7.
45
44
208
186
8.
43.5
59
174
159
9.
44
43
172.5
171
10.
42
41.5
136
138
11.
45
43
128
124
12.
44
46
145
140*
13.
39
39
188
186
14.
43.5
42
121
108
147
122
15.
41
42
120
122
152
139f
16.
50
49
141
124
164
161f
17.
44
47
128
124
155
138
18.
45
45
146
140
166
151
* Total cleavage 76%-79%.
t Total cleavage 85%-86%.
percentage of multipolarity was estimated for the control and experimentally-
treated eggs. Only those batches of eggs were used which on fertilization showed
well-lifted membranes on at least 95 per cent of the eggs. During treatment
with experimental solutions and x-radiation the eggs were at room temperature-,
which varied from 21 degrees to 26 degrees. Fertilization and cleavage were
carried out in a water bath at a temperature of 25.01 ± 0.06 degrees.
The following solutions were used: 0.35 M potassium citrate; CaCl2-sea-water
mixture consisting of two parts of sea water and one part 0.3 M CaCl2; and a
MgCl2-sea- water mixture made up of equal parts of 0.3 M MgCl2 and sea water.
The calcium content of the CaCl2-sea-water mixture is approximately 9.6 times
4 A very few times the small numbers of available eggs made it necessary to make counts
on the living eggs.
RADIOSENSITIV1TY OF ARBACIA EGGS
195
that of sea water. The MgCh-sea- water mixture has a magnesium content 3.3
times that of sea-water. The pH of sea water was 7.9, and the pH of all experi-
mental solutions was 7.6 ± 0.2.
The x-radiation Avas carried out with the dual tube self-rectifying outfit
available at the Marine Biological Laboratory. The secondary voltage was 182
kv., and the current on each tube was 25 ma. The distance from the center of
each target to the center of the material irradiated was 9.5 cm. The eggs were
irradiated in small plastic dishes approximately 2 cm. in diameter. The depth
of the solution containing the eggs was approximately 0.9 cm. Experiments 1
through 8 (Table I) were carried out at an output of 7,600 r per minute, while
all other experiments were exposed at an intensity of 5,600 r per minute.
Viscosity was determined by means of an Emerson hand centrifuge at a
centrifugal force of approximately 1960 X gravity (Wilbur, 1940).
RESULTS
Experiments with Potassium Citrate
Cleavage Time
Unfertilized eggs treated with 0.35 M potassium citrate for 30 and 60 minutes
were given various doses of x-rays and returned to sea water within 30 seconds
following irradiation. The well known effect of roentgen rays in delaying the
TABLE II
Effect of x-radiation on Arbacia eggs treated with potassium citrate for 20 minutes
prior to and 20 minutes following irradiation
Cleavage time of non-irradiated
Cleavage time of eggs receiving
Cleavage time of eggs receiving
eggs
15,200 r
30,400 r
Ex p.
No.
Eggs in
sea water
Eggs treated
with potassium
citrate for
Eggs in
sea water
Eggs treated
with potassium
citrate
Eggs in
sea water
Eggs treated
with potassium
citrate
40 minutes
1.
45 min.
45 min.
82 min.
82 min.
110 min.
103 min.
(for 63% cl.)
(for 63% cl.)
2.
46 min.
46 min.
01
87
128
125
3.
43
42
72
70
126
120
4.
45
47
82
81
125
117
5.
45
45
79
66
138
108
6.
45
45
115
102
170
154
7.
46
46
Exovates on
No exovates.
Exovates on
Exovates
nearly all.
98% cleavage
nearly all.
rare. 100%
Poor cleavage
Poor cleavage
cleavage
8.
43
43
79
78
128
116
cleavage time is shown in Table I. With a dose of 30,400 r the eggs which had
been in potassium citrate for 60 minutes cleaved somewhat sooner than the sea-
water controls in four of the five cases (columns V and VII). With 53,200 r in
12 of the 13 cases studied the citrated eggs cleaved several minutes sooner than
those in sea water (columns VIII, IX and X) ; and the 30-minute citrate treatment
was quite as effective here as the 60-minute treatment. Smaller doses of 3,800
196
K. M. WILBUR AND R. O. RECKNAGEL
and 15,200 r delay cleavage to the same degree in citrated treated eggs and eggs
in sea water (not shown in table).
Eggs treated with potassium citrate for 20 minutes prior to the completion
of irradiation and allowed to remain in citrate for 20 minutes following irradiation
were also protected from the x-ray action to some degree. The effect is clear-cut
with 30,400 r and is indicated in 'some cases at 15,200 r (Table II). Although
a 30-minute treatment with citrate prior to and during x-radiation has little or
no protective action for a dose of 30,400 r (Table I, columns V and VI) a 20-
minute treatment prior to and during x-radiation and followed by an additional
130
120
(/) HO
o
o
(f)
u
UJ
Qiao
70
6O
50.
15 30 45 60
IMMERSION TIME-MINUTES
FIGURE 1. Viscosity of unfertilized Arbacia eggs in 0.35 M potassium citrate. The relative
viscosity (ordinates) was measured following treatment in potassium citrate for various periods.
(abscissas). pH 7.6 Temperature 24.0-25.2° C.
20-minute immersion after irradiation may inhibit the x-ray action. The
difference is not especially striking, and we should not care to stress the point
on the basis of the evidence at hand. However, the data do suggest the inter-
esting possibility that the x-ray effect can be inhibited somewhat by changing
the ionic composition of the protoplasm following the period of irradiation.
When eggs are x-rayed in sea water and immersed in potassium citrate
immediately afterward, the citrate has no protective action. Five such experi-
ments were carried out in which eggs were given doses of 15,200 and 30,400 r
and changed from sea water to citrate in less than 30 seconds following irradiation,
and immersed for 30 minutes. In this case some time would be required for
equilibrium to be established between the citrate and the egg; and reactions
RADIOSENSITIVITY OF ARBACIA EGGS 197
initiated by the radiation may have gone to completion before the citrate exerted
its full effect.5
Viscosity
The effect of 0.35 M potassium citrate on the colloidal state of the Arbacia
egg at the time of irradiation as reflected in its viscosity has been studied. The
viscosity changes of eight batches of eggs have been determined, and the results
for four of these are shown in Figure 1. It is to be noted that potassium citrate
causes an increase in viscosity. The highest value is usually reached in 30
minutes and maintained constant with continued immersion. Our concern has
not been with the mechanism of the viscosity increase produced by potassium
citrate. However, it may be pointed out that the potassium ion will in itself
increase the viscosity of protoplasm (Heilbrunn, 1937). Mazia (1940) has found
a marked decrease in the calcium content of Arbacia eggs treated with potassium
citrate; and this has been confirmed by Miss Pauline Hamilton for the particular
conditions of our experiments.
Experiments with Magnesium Chloride
Experiments similar to those with potassium citrate were carried out with a
mixture of equal parts of 0.3 M MgCla and sea water. The total period of
immersion in the experimental solution was 60 minutes. The response to
x-radiation of eggs treated with this mixture was much the same as in sea water.
In each of six experiments doses of 15,200 r and 30,400 r were used. A dose of
53,200 r was employed in four experiments.
Viscosity determinations on eggs immersed for 55 minutes in the MgCl2-sea-
water mixtures revealed a slight decrease in seven of nine batches of eggs. The
average decrease in viscosity for these seven samples was approximately 12
per cent.
Experiments with Calcium Chloride
The effects of x-radiation on eggs treated for 60 minutes with a mixture of
one part 0.3 M CaCl2 and two parts-sea water were similar to those produced on
eggs irradiated in sea water. Doses of 3,800 r, 15,200 r, 30,400 r and 53,200 r
were used.
The CaCl2-sea-water mixture resembles MgCl2-sea- water mixture causing a
slight decrease in the viscosity of unfertilized eggs. The average decrease for
five batches of eggs was about 15 per cent after 60 minutes treatment.
The Viscosity of Unfertilized Eggs Following X-Radiation
In collaboration with Mr. Walter Wilson the viscosity of unfertilized Arbacia
eggs has been studied after irradiation in sea water in order to ascertain whether
roentgen rays will produce viscosity changes in the living cell. A dose of 30,400 r
was employed and the viscosity determined 25 minutes following the completion
5 Such an assumption, however, involves an apparent contradiction in that the possible
enhanced effect resulting from leaving eggs in citrate for a 20-minute period following irradiation
would argue that the x-ray effect was not complete shortly after irradiation. But the situation
in which sea water replaces citrate is not necessarily comparable to the present one in which
citrate replaces sea water.
198 K. M. WILBUR AND R. O. RECKNAGEL
of the irradiation or approximately 30^ minutes from the time that irradiation was
begun. The viscosity determinations were carried out at 24.4-25.8° C. This
dosage has a drastic effect upon cleavage. The average cleavage time for 21 ex-
periments was 134 minutes as compared with 44 minutes for the non-irradiated
control eggs. The majority of eggs receiving this dosage also exhibit multipolar
cleavage. However, this relatively enormous dose failed to produce detectable
changes in the viscosity of the egg (five experiments).
The centrifuge method as used here would enable one to distinguish between
a relative viscosity of 70 units and one of 60 units, for example. Our negative
results therefore apply only to differences of this order of magnitude.
We are indebted to Dr. L. V. Heilbrunn for his co-operation and advice;
to Miss Pauline Hamilton for her kindness in carrying out the calcium analyses;
and to Dr. F. R. Hayes for helpful suggestions during the preparation of the
manuscript.
DISCUSSION
The data presented indicate that potassium citrate inhibits the effect of
x-radiation on cell division. However, the inhibition is slight and appears only
with high x-ray doses. The effect of the citrate treatment prior to irradiation
is to increase the viscosity of the protoplasm and to reduce the calcium content.
But it is also almost certainly true that immersion of a cell in potassium citrate
upsets the entire ionic equilibrium of the cell and not merely the calcium. content.
In view of this, the influence of the potassium citrate treatment may involve
substances other than calcium. Dale (1942) found that various substances,
including sodium oxalate, sodium nitrate and sodium nitrite, would inhibit the
destruction of d-amino-acid oxidase by x-rays. As yet, however, there is no
justification for assuming enzyme inhibition by citrate in the case of the Arbacia
egg-
The experiments with calcium-rich and magnesium-rich sea water together
with the citrate experiments at lower x-ray doses indicate that the egg probably
can tolerate a considerable change in ionic composition without an alteration in
radiosensitivity. That the colloidal state of the protoplasm was affected by the
addition of these ions is shown in most cases by a change in the viscosity which
is increased by potassium citrate and decreased by sea water containing excess
calcium or magnesium.
Experiments were described pointing to a possible action of potassium citrate
after the period of irradiation. Even in those cases in which eggs were changed
from citrate to sea water immediately following irradiation, some time would be
required before equilibrium could be established. It may be true that the entire
action of citrate is exerted after irradiation. If such is the case, one would have
to assume that at least a portion of the x-ray action is indirect. That is, the
x-radiation initiates a reaction which is partially inhibited in the citrated egg.
It is rather remarkable that the viscosity of the unfertilized egg is unchanged
by doses of radiation which so greatly alter the rate and normal course of cell
division. The direct coagulation of proteins as an explanation of the biological
effects of roentgen rays would seem to be ruled out in the present study (see
Zirkle, 1940).
RADIOSENSITIVITY OF ARBACJA EC
JS 199
We should like to suggest that the chief action of x-£ys on th(p egg is the
alteration of some system, perhaps enzymic, which comes inP Prominence after
fertilization and is of particular importance for certain phases ot T^tosis. This
explanation has also been suggested for colchicine which may be wiuQut effect
on the viscosity of the unfertilized Arbacia egg, yet changes the viscosity of^be
fertilized egg and inhibits cell division (Wilbur, 1940). That radiation ma>
interfere with cellular respiratory systems has been pointed out by several
workers (see, for example, Crabtree and Cramer, 1933; Rudisill and Hoch, 1938).
We may call attention to the interesting fact that eggs can be treated for
relatively long periods with isotonic potassium citrate or solutions of high calcium
or magnesium content and yet on return to sea water they can be fertilized and
will usually cleave at a normal rate. The citrate and magnesium treatments may,
however, cause a slight amount of multipolarity.
r
SUMMARY
1. Treatment of Arbacia eggs with 0.35 M potassium citrate inhibited the
retarding action of x-radiation on cell division. However, the inhibition by
citrate was slight and appeared mainly with high x-ray doses (30,400 and 53,200 r).
2. The radiosensitivity of the egg was unaffected by increasing the calcium
or magnesium content of the sea- water medium.
3. The potassium citrate treatment employed increased the viscosity of the
unfertilized egg. The viscosity was decreased slightly in the sea-water solutions
of increased calcium or magnesium content.
The data presented indicate that changes in the ionic composition and
viscosity of the protoplasm may occur without altering the sensitivity of the egg
to x-radiation.
4. Doses of x-radiation which markedly altered the rate and normal course
of cell division produced no detectable change in the viscosity of the unfertilized
egg-
LITERATURE CITED
ANDERSON, R. S., AND H. TURKOWITZ, 1941. The experimental modification of the sensitivity
of yeast to roentgen rays. Amer. Jour. Roent., 46: 537-541.
CRABTREE, H. G., AND W. CRAMER, 1933. The action of radium on cancer cells. II. Some
factors determining the susceptibility of cancer cells to radium. Proc. Roy. Soc. London,
B, 113: 238-250.
DALE, W. M., 1942. The effect of x-rays on the conjugated protein d-amino-acid oxidase.
Biochem. Jour., 36: 80-85.
EVANS, T. C., J. C. SLAUGHTER, E. P. LITTLE, AND G. FAILLA, 1941. The influence of the medium
on the radiosensitivity of sperm. Biol. Bull., 81: 291-292.
HEILBRUNN, L. V., 1937. An Outline of General Physiology. W. B. Saunders Co., Philadelphia:
76-77.
HEILBRUNN, L. V., AND K. M. WILBUR, 1937. Stimulation and nuclear breakdown in the Nereis
egg. Biol. Bull., 73: 557-564.
MARSHAK, A., 1938. Alteration of chromosome sensitivity to x-rays with NH4OH. Proc. Soc.
Exp. Biol. Med., 38: 705-713.
MAZIA, D., 1940. The binding of ions by the cell surface. Cold Spring Harbor Sympos., 8:
195-203.
MOTTRAM, J. C., 1935. On the alteration in the sensitivity of cells towards radiation produced
by cold and by anaerobiosis. Brit. Jour. Radiol., 8: 32-39.
RUDISILL, H., AND J. H. HOCH, 1938. How x-rays may kill cells. Radiol., 31: 104-106.
200
f. WILBUR AND R. O. RECKNAGEL
SCOTT, C. M., 1937. Sonie quantitative aspects of the biological action of x and 7 rays. Med.
Res. Counc. Spec. Rep. Ser., No. 223.
WILBUR, K. M., J939. The relation of the magnesium ion to ultra-violet stimulation in the
Nereis egg. Physiol. Zool., 12: 102-109.
WILBUP. K. M., 1940. Effects of colchicine upon viscosity of the Arbacia egg. Proc. Soc. Exp.
' Biol. Med., 45: 696-700.
ZIRKLE, R. E., 1936. Modification of radiosensitivity by means of readily penetrating acids
and bases. Amer. Jour. Roent., 35: 230-237.
ZIRKLE, R. E., 1940. The influence of intracellular acidity on the radiosensitivity of various
organisms. Jour. Cell. Comp. Physiol., 16: 301-311.
ZIRKLE, R. E., 1941. Combined influence of x-ray intensity and intracellular acidity on radio-
sensitivity. Jour. Cell. Comp. Physiol., 17: 65-70.
THE DISTRIBUTION AND REPRODUCTION OF SAGITTA ELEGANS
ON GEORGES BANK IN RELATION TO THE
HYDROGRAPHICAL CONDITIONS
GEORGE L. CLARKE, E. LOWE PIERCE, AND DEAN F. BUMPUS
(Biological Laboratories, Harvard University and Woods Hole Oceanographic Institution) l
During the past few years an investigation has been undertaken of the factors
underlying the productivity of Georges Bank, an extensive fishing area lying
east of Cape Cod (Fig. 1). The economy of this area depends upon various
interdependencies of the fish populations, the bottom fauna, and the plankton;
and all are profoundly affected by the complex of strong currents and persistent
eddies which are found on the Bank. In order to understand the essential
ecological relationships, it is therefore necessary to gain a knowledge of both the
hydrography and the biology of the waters of the region.
The present study of the abundance, distribution, and seasonal cycle of
reproduction of the chaetognath, Sagitta elegans, was undertaken first because
this species forms a prominent element in the zooplankton of Georges Bank, and
second because Sagitta may be used as a "current indicator" to aid in unravelling
the involved current system of the region. This species is a relatively large,
easily recognized member of the zooplankton and its body length and maturity
stage are readily determined. The life span of Sagitta elegans is sufficient to
bridge periods of six weeks or more, with the result that in cases where observa-
tions are repeated each month, the same population may be identified from one
cruise to the next. This condition presents a desirable contrast to more rapidly
reproducing organisms, such as diatoms, in which populations of large dimensions
may appear or disappear within a week or so.
Certain relatively recent investigations of the ecology of Sagitta in other
regions are available for comparison, but none had the advantage of our quanti-
tative collection method, nor the opportunity for revisiting as frequently over a
two-year period an extensive net-work of stations as characterized the present
undertaking. The breeding and growth of Sagitta elegans was studied by Russell
(1932; 1933) off Plymouth, England, and by Pierce (1941) in parts of the Irish
Sea. Sagitta elegans has been employed successfully as a current indicator in
British waters by Russell (1939) and the distribution of the species by currents
in the Gulf of Maine has been critically investigated by Redfield and Beale (1940).
COLLECTION AND ANALYSIS OF MATERIAL
Samples of plankton and hydrographic data for the present study were
obtained from the research vessel, "Atlantis", during eleven cruises to Georges
Bank from September 1939 to June 1941 (Table I). On each cruise a net-work
of 21 to 52 stations was occupied over the Bank. In all cruises (except that of
1 Contribution No. 328.
\
201
202
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
January, 1940) the stations were ordinarily placed at 15-mile intervals on five
or six parallel sections, about 25 miles apart, running SE and NW across the
Bank and into the immediately adjacent waters. The location of the stations
is indicated in the charts showing the distribution of Sagitta (Figs. 5 and 6).
The stations covered the region from South Channel on the southwest to the
eastern tip of Georges Bank and from the deep basin of the Gulf of Maine on
the northwest to the edge of the continental shelf on the southeast. No stations
TABLE I
List of cruises to Georges Bank
Cruise no.
Date
No. of stations
Station serial nos.
89
Sept. 6-13, 1939
52
3629-3680
93
Jan. 4-11, 1940
21
3726-3746
95
Mar. 21-Apr. 2, 1940
35
3792-3826
96
Apr. 17-27, 1940
26
3827-3852
97
May 9-16, 1940
33
3856-3888
98
June 1-8, 1940
36
3892-3927
100
June 19-27, 1940
36
3932-3967
112
Mar. 21-Apr. 2, 1941
33
4177-4209
113
Apr. 15-23, 1941
34
4210-4243
114
May 7-14/1941
34
4244-4277
116
May 28-June 4, 1941
33
4278-4311
could be made in the immediate vicinity of Cultivator and Georges Shoals.
The segment of the ocean covered by the station net-work of each cruise was
more than 150 miles long and 100 miles wide, or an area larger than the states
of Massachusetts, Connecticut, and Rhode Island combined (Fig. 1).
Standard hydrographic observations for salinity and temperature were made
at every station and Secchi disc measurements of transparency were carried
out during daylight stations. Studies of certain chemical characteristics of the
water and of the phytoplankton population were undertaken by collaborating
investigators (Sears, 1941; and Riley, 1941 and 1942).
The zooplankton was collected at each station by means of two or more hauls
with Plankton Samplers (Clarke and Bumpus, 1940) and one haul with a stramin
net. Sagittae were taken in adequate numbers in both types of equipment and
the two sets of hauls served as a check on one another.
The opening of the Plankton Sampler, which is 12.7 cm. in diameter, is
provided with a shutter, and each instrument contains a meter which records the
amount of water filtered by the net. In the present case, the instruments were
equipped with No. 2 silk nets (22 strands/cm.) and "oblique" hauls l were made
at a speed of about 2 knots for periods of 25 to 40 minutes. Ordinarily between
10 and 20 cubic meters of water were filtered during each tow, but the action of
1 In an "oblique" haul the net is towed horizontally but is raised in steps so that the whole
depth of the stratum concerned is sampled. The Sampler could be towed safely down to within
three meters of the actual bottom.
SAGITTA ELEGANS ON GEORGES BANK
203
the tide or of clogging was such that values as low as 5 m.3 and higher than
30 m.3 were recorded. This variation makes clear the need for measuring the
amount of water which actually passes through the net. The Samplers were
arranged vertically so as to divide the total depth of water into two or three
strata and, when feasible, were attached to the same cable. The uppermost
FIGURE 1. Orientation map of Atlantic Coast. The location and relative size
of Georges Bank are indicated.
instrument sampled the "Shallow" Stratum, extending from a depth of 25 m. to
the surface. The lower limit of this stratum corresponded roughly with the
position of the thermocline in those areas where it existed. At stations where
the water was less than 75 m. deep, the "Second-depth" Stratum extended from
the bottom to 25 m. In water deeper than 75 m., however, the remaining
distance to the bottom (or to a maximum depth of 200 m.) was divided into two
equal parts and these comprised the "Second-depth" and the "Deep" Strata
204
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
respectively. The vertical distribution of the sagittae could therefore be studied
on the basis of these strata :
Stratum
"Shallow"
"Second-depth"
"Deep"
Water less than 75 m.
0 m. to 25 m.
25 m. to bottom
Water more than 75 m.
0 m. to 25 m.
25 m. to half distance to bottom (or to half distance to
200 m.)
Remaining distance to bottom (or to 200 m.)
The stramin net (Diameter: 1.5 m., Mesh: 6 strands/cm.) was equipped with
rollers at the lower edge of its frame in order that it could be safely lowered until
it touched the bottom. One "oblique" haul was made from the bottom (or
from a depth of 200 m.) to the surface at each station. When proper allowance
o/
/c
o
•z.
LU
O
LU
rr
30
20-
10-
20J
SEPT
PLANKTON SAMPLER
STRAMIN NET
75CM SILK NET
JAN
12 16 20 24
LENGTH
28 MM
FIGURE 2. Comparison of length frequency distribution of Sagitla elegans for the following
types of nets:
Plankton Sampler (12.7 cm. in diameter) with No. 2 silk.
Silk net (75 cm. in diameter) with No. 2 Silk.
Stramin net (1.5 m. in diameter).
was made for the difference in the sizes of the apertures of the stramin net and
the Plankton Samplers, a good agreement was found between the numbers of
sagittae taken by the former and the sum of the catches of the latter at each
station.
Before the work was begun, it was doubted whether the relatively small
Plankton Sampler would catch the larger sizes of an active animal, such as
Sagitta, in their true proportions. For the first cruise (September 1939), there-
fore, a silk net 75 cm. in diameter and of the same mesh, was towed immediately
below the Plankton Sampler. Since comparison of the length frequency distribu-
tion of the sagittae taken by the two sizes of nets showed exceptionally good
agreement (Fig. 2), it is felt that the catch of the Plankton Sampler can be relied
upon. In the January cruise sagittae as large as 30 mm. in length were retained
SAGITTA ELEGANS ON GEORGES BANK 205
by the Sampler. On the other hand, the stramin net was shown not to retain
adequately the smallest sizes of sagittae. For these reasons and especially
because of the accuracy of the determinations of depth and volume with the
Plankton Samplers, the ensuing analysis of the abundance and distribution of the
Sagitta populations is based primarily upon the hauls with these instruments.
The present observations can therefore be placed on a quantitative basis not
hitherto possible.
The sagittae were separated from the remaining plankton in the laboratory
and the species present were identified and enumerated.1 The great majority of
sagittae were Sagitta elegans but specimens of 5. serratodentata and a smaller
number of S. enflata were encountered in certain hauls from the periphery of
Georges Bank. For each station the average number of 5. elegans per cubic
meter was calculated for each stratum by dividing the number caught in each haul
by the volume of water filtered by the Plankton Sampler. The total number of
individuals under each square meter of sea surface was found by multiplying the
number per cubic meter for each stratum by the thickness of the appropriate
stratum and then adding these products together. Finally the average number of
animals per cubic meter for the whole water column at each station was obtained
by dividing the foregoing value by the total depth of water at each station.
These average values per cubic meter (or per ten cubic meters) have been plotted
on the charts showing quantitative distribution (Figs. 5, 6, and 10), but they
may readily be re-converted to the "per square meter" basis by multiplying by
the depth in each case.
Length measurements were made of all specimens of Sagitta elegans in each
haul up to a maximum of 50. The stage of maturity was also determined for
the individuals of this species in most of the hauls in each cruise from all parts
of the Bank. Animals from each haul (usually between 20 and 50 individuals)
were stained by the method described by Pierce (1941, p. 115), and then were
classified as Stage I — Immature, Stage II — Intermediate, or Stage III — Mature,
following the criteria of Russell (1932, p. 134).
GENERAL HYDROGRAPHICAL CONDITIONS
The depth of the major portion of Georges Bank lies between 40 m. and
100 m., although areas of less than 25 m. occur in the north central portion, and
the Shoals themselves are covered by only 5 to 15 m. of water. Along the
northern edge of the Bank the bottom drops rapidly from about 40 m. to more
than 200 m. as the deep basin of the Gulf of Maine is approached. Along the
southern edge the depth changes somewhat more gradually from 100 m. to 200 m.
Beyond 200 m. it increases rapidly to about 2000 m.
Georges Bank is therefore, roughly speaking, a submerged, flat-topped plateau
(Fig. 3), and it presents a sufficiently large obstacle to water movement to produce
a profound effect on the ocean currents of this region. Although the details of
the water movements over and around the Bank have never been adequately
determined, especially for the colder part of the year, it has been well established
in general that during the summer months at least, water from the Gulf of Maine
1 The authors are indebted to Miss Dorcas Delabarre for technical assistance in the analysis
oc the Sagitta material.
206
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
does not flow directly across the Bank but tends to move around the eastern
and southern margins of the Bank in a clockwise direction, leaving a relatively
stationary eddy of water over the central part of the Bank. From the point
of view of the ecology of the Bank, our interest in the current system lies in the
question of the degree of permanence of this eddy, and in the extent to which
the "bank water" can be regarded as biologically isolated from the surrounding
regions.
The eddy on the Bank might be dislodged by relatively slight changes in the
strength or position of the surrounding ocean currents (Iselin, 1939), or it might
be disrupted by the action of certain local agents. The strong tidal currents on
FIGURE 3. Vertical sections for temperature and salinity for September 1939 (cruise 89).
The contour of Georges Bank is indicated by the cross hatching from the Gulf of Maine on the
left to the edge of the Continental Shelf on the right. Station numbers appear at the top of the
diagrams. The figures on the salinity curves are to be increased by 30 to give the actual values
in parts per mille.
the Bank cause the overlying water to oscillate in generally elliptical paths, the
long diameters of which may exceed eight miles. Winds, which frequently reach
gale velocities, sweep unimpeded across the area, tending to force the surface
water along with them. The danger would thus appear to exist that from time
to time the bank water might be swept entirely off the Bank, carrying with it
the pelagic stages of animals which, as adults, could live only in a bank environ-
ment, or removing an element of the plankton which is essential to the economy
of the Bank. Even though no cataclysmic dislocation of the bank water occurred,
it is important to know to what extent a dilution or a renewal of the water mass
may take place through continuous or intermittent admixtures of new water from
one direction or another.
The turbulence produced by the tidal currents and by the wind in the rel. -
tively shallow water overlying Georges Bank causes a vertical mixing of the
SAG1TTA ELEGANS ON GEORGES BANK
207
water which results in a nearly uniform distribution of temperature and salinity
from top to bottom at all seasons of the year, particularly in the central part of
the Bank. The bank water thus contrasts sharply with the surrounding water
masses, which are typically stratified during all except the winter months. Since
the temperatures and salinity values on the Bank are generally intermediate
between those of the surface and the deeper strata on the Gulf of Maine but
usually much lower than those of the water lying to the south, we know that the
bank water is originally derived, in a large part at least, from the Gulf (Figs. 3
and 4). That portion of the Bank over which vertically uniform water was
found is termed the Mixed Area, and all stations at which the salinity does not
vary by more than 0.2 part per mille from surface to bottom are considered to
WESTERN SECTION
TEMPERATURE °C
FIGURE 4. Vertical sections for temperature and salinity for May 1940 (cruise 97).
lie within it.1 The limits of the Mixed Area are ordinarily rather sharp, and have
been indicated by a heavy broken line in the charts of Sagitta distribution for
each cruise (Figs. 5, 6, and 10).
The vertical uniformity of the temperature and salinity within the Mixed
Area presents an ecological condition for the Bank organisms which is quite
unlike that for the oceanic forms living in the stratified water of the adjacent
deeper areas. Moreover, seasonal changes in these factors are somewhat damped
by the continued vertical mixing, as is seen by reference to Tables II and III.
The seasonal range in temperature for the Mixed Area extended from a minimum
of about 2.5° C. to a maximum of over 16° C. Surface temperatures in the
regions to the north and to the south of Georges Bank were generally higher in
summer and lower in winter. Furthermore, during the winter pelagic animals
living at the surface in these neighboring areas could find warmer water by
descending to lower strata, and similarly during the summer they could escape
excessively high temperatures by seeking greater depths. In contrast, the fauna
1 Uniformity of salinity, rather than temperature, was taken as the criterion for the Mixed
Area because in the present situation salinity is less easily modified after the water has reached
the Bank.
208
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
TABLE II
Comparison of temperatures in the Mixed Area and in the Stratified Water
at the margins of Georges Bank
Values given are for typical stations near the center of the Bank and in the deeper water to
the north and to the south. At the stations in the Mixed Area temperatures did not vary by
more than 1.6° C. from surface to bottom and in most cases the variation was much less.
Mixed Area
North Margin
South Margin
Date
Aver. temp.
Surface
100 m.
Surface
100 m.
Sept., 1939
16.3° C.
16.4° C.
6.3° C.
19.3° C.
9.3° C.*
Jan., 1940
4.1
3.3
6.8
4.6
6.2*
Mar., 1940
2.5
2.1
5.3
2.8
3.9*
Apr., 1940
3.7
3.5
3.4
4.7
11.2
May, 1940
4.8
5.3
4.1*
6.1
7.8*
June 1-8, 1940
7.2
10.4
3.0*
10.8
9.3
June 19-27, 1940
8.5
10.6
3.1
10.3
10.2
Mar., 1941
2.7
3.3
4.1
2.9
4.9
Apr., 1941
3.9
4.3
4.5
3.2
4.9
May, 1941
4.6
6.5
5.4
4.0
4.5
June, 1941
6.8
9.1
3.6
7.3
5.4
* Value at somewhat less than 100 m.
TABLE III
Comparison of salinities in the Mixed Area and in the Stratified Water
at the margins of Georges Bank
Values given are for typical stations near the center of the Bank and in the deeper water to
the north and to the south. At the stations in the Mixed Area salinities did not vary by more
than 0.2°/oo from surface to bottom.
Mixed Area
North Margin
South Margin
Date
Aver, salinity
Surface
100 m.
Surface
100 m.
Sept., 1939
32.5°/oo
32. l°/oo
32.5°/oo
33.4%0
33.8°/oo*
Jan., 1940
32.8
31.5
33.7
32.9
33.2*
Mar., 1940
32.8
32.4
33.2
32.7
33.0*
Apr., 1940
33.0
32.5
33.1
33.0
35.3
Mav, 1940
32.8
32.5
33.2*
32.7
34.1*
June 1-8, 1940
32.8
31.8
32.9*
33.1
33.5*
June 19-27, 1940
32.7
32.0
32.9
32.5
34.4
Mar., 1941
32.7
32.8
33.1
32.7
33.4
Apr., 1941
32.5
32.5
33.2
32.1
33.5
May, 1941
32.5
32.3
33.4
32.1
33.4
June, 1941
32.7
32.3
32.8
32.6
33.5
* Value at somewhat less than 100 m.
SAGITTA ELEGANS ON GEORGES BANK 209
of the central bank waters could reach a materially different temperature only
by migrating laterally entirely out of the Mixed Area. A similar situation
obtains in regard to salinity, although there is little evidence that changes in
salinity, per se, of the magnitude encountered in this region are of ecological
importance. On the other hand, differences in density, which result in large
part from the salinity, are bound to be critical for passively floating organisms,
and the lack of a pronounced vertical density gradient in the Mixed Area, as well
as the excessive turbulence there, presents a very special problem for such forms.
QUANTITATIVE DISTRIBUTION OF SAGITTA ELEGANS
Vertical Distribution
The numerical abundance of Sagitta elegans varied greatly among individual
hauls, ranging from a maximum of 165 specimens per cubic meter "• to zero.
When the hauls of each cruise are considered together, however, certain general
trends in the changes in the Sagitta population become clear. The changes in
horizontal distribution from cruise to cruise will be presented in the next section.
In this section the variations in the vertical distribution will be considered as
derived at each station from the separate hauls with the closing Plankton Samplers
for the Shallow Stratum, the Second-depth Stratum, and the Deep Stratum.
At stations where the depth of water was less than 75 m. the Second-depth haul
extended to the bottom. Since this situation obtained at the majority of stations
within the Mixed Area, the chief comparison for vertical distribution is between
the Shallow Stratum and the Second-depth Stratum.
It is obvious that vertical distribution at stations made during the day
(between the hours of sunrise and sunset) had to be distinguished from the
situation at stations made during the night, since a diurnal migration of the
animals was to be expected (Russell, 1933). Furthermore, if a vertical migration
of the Sagitta tended to take place, very different conditions would be met with
according to whether the station was in the Mixed Area or in the Stratified Area.
If the animals encountered a thermocline, their movement might be stopped,
or reversed (cf. Clarke, 1934). For the foregoing reasons the hauls upon which
the analysis of the vertical distribution of Sagitta elegans is based, have been
subdivided into those made at stations in the Mixed Area and those made at
stations in the Stratified Area and have been further subdivided in each case
into day and night hauls.
The average abundance of Sagitta elegans for all stations in each of these
categories varied considerably from cruise to cruise (Table IV). In September
the larger number of animals was found in the Second-depth Stratum in all cases,
although at night in the Mixed Area an almost equally great number was taken
at the upper level. In the winter and early spring of 1940 much smaller average
numbers of sagittae were encountered and the differences in the various strata
were not large. A tendency for the largest hauls to occur in the Deep Stratum
is to be noted for May 1940, but this generality does not hold for the more
sizable catches of the June cruises of that year. During the early spring of
1941 small numbers of sagittae were again encountered and their vertical distri-
1 Shallow stratum haul, May 30, 1941, made in south central part of Bank and consisting of
very small individuals.
210
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
TABLE IV
Numerical abundance of S. elegans in the separate strata. Average number per cubic meter in the
indicated categories.
X indicates an abundance of less than 0.1/m3. Values placed in parentheses are based on a
total of less than 5 hauls.
Mixed Area
Stratified Area
Month
Cruise
Stratum
Day
Night
Day
Night
Sept., 1939
89
Shallow
5.3
17.5
0.7
1.6
2nd Depth
10.8
17.9
8.1
(8.5)
Jan., 1940
93
Shallow
2.4
6.8
0
2nd Depth
3.8
7.6
X
Deep
(0.5)
(0)
0
Mar., 1940
95
Shallow
2.4
1.8
0
X
2nd Depth
2.4
1.1
0
0.1
Deep
(1.9)
(0.2)
X
X
Apr., 1940
96
Shallow
1.8
2.2
X
(0)
2nd Depth
3.6
1.4
0.2
(0)
Deep
(2.8)
(0.3)
(0)
May, 1940
97
Shallow
2.5
5.1
0.2
0.6
2nd Depth
6.2
5.1
0.2
0.3
Deep
(9.0)
(1.0)
16.6
13.0
June 1-8, 1940
98
Shallow
16.9
3.3
4.5
(3.3)
2nd Depth
40.4
50.1
3.8
(5.9)
Deep
(13.1)
(25.0)
3.6 '
(8.7)
June 19-27, 1940
100
Shallow
35.6
20.8
5.3
17.4
2nd Depth
58.6
44.5
1.8
9.5
Deep
(58.4)
3.4
12.6
Mar., 1941
112
Shallow
2.4
2.2
0
0.8
2nd Depth
2.5
2.8
0
0.8
Deep
(1.1)
(1.2)
X
1.2
Apr., 1941
113
Shallow
2.1
0.8
0
0.4
2nd Depth
3.2
1.9
0
0.9
Deep
(1.3)
(0.1)
0.1
0.1
May, 1941
114
Shallow
1.4
0.6
0
0.3
2nd Depth
3.5
0.6
X
X
Deep
(0.6)
(0)
0.3
1.0
June, 1941
116
Shallow
46.9
14.6
2.5
0.7
2nd Depth
45.6
17.8
1.4
1.9
Deep
(3.4)
(45.8)
1.6
2.5
Averages
Shallow
10.9
6.9
1.3
2.3
2nd Depth
16.4
13.7
1.6
2.5
Deep
3.7
14.6
2.9
3.9
bution was found to be generally uniform. This situation also held for May of
that year, but in June much larger catches were made especially in the two
upper strata for the day hauls and for the Deep Stratum for the night hauls in
the Mixed Area.
SAGITTA ELEGANS ON GEORGES BANK
211
In order to ascertain what tendency existed toward vertical diurnal migration
it is not satisfactory to employ the foregoing average values because of the
likelihood that a few large hauls would obscure differences occurring in stations
with smaller representation. Accordingly a calculation has been made of the
percentage of stations in each category for each cruise at which the number of
Sagitta elegans in the Second-depth haul was greater than in the Shallow haul
(Table V). When the data are scrutinized on this basis, it becomes clear that a
TABLE V
Comparative vertical distribution of S. elegans for day and night hauls
Percentage of Stations at which the number of animals per cubic meter in the "Second-
Depth" haul was greater than in the "Shallow" haul. Values placed in parentheses are based on
a total of less than 5 cases.
Mixed Area
Stratified Area
Month
Cruise
Day
Night
Day
Night
Sept., 1939
89
100%
29%
93%
50%
Jan., 1940
93
60
57
~(100)
Mar., 1940
95
64
43
(67)
Apr., 1940
96
71
25
(100)
May, 1940
97
86
50
60
(0)
June 1-8, 1940
98
70
71
(67)
(67)
June 19-27, 1940
100
75
100
20
33
Mar., 1941
112
78
70
(50)
Apr., 1941
113
73
(75)
(100)
May, 1941
114
75
(50)
(100)
(100)
June, 1941.
116
46
80
(25)
50
Averages
73%
59%
66%
62%
definite vertical migration was taking place in both the Mixed Area and the
Stratified Area at the time of the September cruise, since the majority of the
animals were found below 25 m. in the day time and above 25 m. at night. A
similar tendency, but less marked, was encountered in the first four cruises of
1940 and in the May cruise of 1941 for the Mixed Area. A reversal of the
situation is to be noted for the June cruises in both 1940 and 1941, for in those
cases the Second-depth hauls were greater at night at more stations than during
the day. 'Taking the average for all cruises it is apparent that in all situations
the deeper hauls were numerically greater in more than 50% of the cases. How-
ever, the variations encountered in vertical distribution and in diurnal migration
from cruise to cruise show that the reactions of Sagitta in maintaining its vertical
position in the water change materially according to the season or in relation to
size a d stage of maturity. A similar conclusion was reached by Russell (1933).
212
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
Horizontal Distribution Throughout the Year
The quantitative aspects of the distribution of Sagitta elegans will be examined
in relation to the location of the water masses on Georges Bank for September
FIGURE 5. Distribution of Sagitta elegans on Georges Bank during 1940. January (cruise
93), March (cruise 95), April (cruise 96), May (cruise 97), June 1-8 (cruise 98), and June 19-27
(cruise 100). Average numbers per 10 cubic meters for whole water column for ail stages.
Plankton Sampler hauls. Boundary of Mixed Area indicated by heavy broken line.
1939 (Fig. 10), for the winter and spring of 1940 (Fig. 5), and for the spring of
1941 (Fig. 6). In each chart the station positions are designated by blar k dots
and the average number of sagittae per 10 cubic meters for the whole water :olumn
SAGITTA ELEGANS ON GEORGES BANK
213
at each station is indicated.1 Contour lines representing concentrations of 1, 10,
(50), 100, (500) and 1000 individuals per cubic meter have been drawn in. Pro-
gressively dense cross-hatching indicates areas of increasing numerical abundance.
In addition, the position of the margin of the vertically homogeneous water of
the Mixed Area for each cruise has been indicated by a heavy broken line super-
imposed independently on each chart.
FIGURE 6. Distribution of Sagitta elegans on Georges Bank during 1941. March (cruise
112), April (cruise 113), May (cruise 114), and June (cruise 116). Average numbers per 10 cubic
meters for whole water column for all stages. Plankton Sampler hauls. Boundary of Mixed
Area indicated by heavy broken line.
Inspection of the chart for September 1939 (Figure 10, upper left) reveals
the fact that at the time of this cruise extremely small numbers of Sagitta elegans
occurred around the margins of the Bank (except possibly in the north central
region). It is clear that the area of greatest concentration of this species lay in
the center of the Bank. During this period the Mixed Area did not cover the
whole of Georges Bank but was confined chiefly to the central portion except in
the north where it extended beyond depths of 100 m. A rough correspondence
1 The total number of sagittae under each square meter of sea surface may be derived from
these values by multiplying by the depth (see "Collection and Analysis of Material")-
214 G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
is seen to exist for this cruise between the contour for 100 sagittae per 10 cubic
meters (or 10 animals per cubic meter) and the limits of the Mixed Area.
A similar scrutiny of the distribution of Sagitta elegans may be undertaken
for the succeeding cruises from Figures 5 and 6. A great variation in the nu-
merical strength of this species during the year is indicated by the fact that
although numbers greater than 10/m.3 were encountered at nine stations in the
September cruise, only one to four stations were as rich during the next four
cruises. However, in each of the June cruises of 1940 about 20 stations yielded
an average abundance of more than 10/m.3 and one station of more than 100/m.3
Similarly in 1941 during the first three cruises there were no hauls containing
more than 10/m.3 but in the June cruise this value was exceeded at 18 stations
and there was one instance of an abundance greater than 100/m.3 Although it
was unfortunately not possible to make observations in every month of the year,
as would have been desirable, the available information strongly indicates that
the numerical strength of Sagitta elegans in the Georges Bank region reaches a
low ebb in the winter and early spring, and attains high values beginning in
June and perhaps extending through the summer.
In each of the eleven cruises the center of abundance of Sagitta elegans was
found to be located within the central portion of the Bank and numbers dropped
off toward the margin. Along the southern edge of the Bank the concentration
of this species dwindled to a very small proportion and frequently to zero,
especially beyond the 200 m. contour. Similarly low numbers were usually
encountered along the eastern and northern margins although in some cruises an
insufficient number of stations was occupied beyond the Bank to make certain
of the limits of the population to the north. Since in most cases Sagitta elegans
occurred in abundance at the westernmost stations of each cruise, we have
definite indication that, at certain seasons of the year at least, numbers of this
species are transported by westerly currents across South Channel toward
Nantucket Shoals.
When the center of abundance of Sagitta elegans is compared with the location
of the Mixed Area, it is clear that in spite of the changes in position of the latter
from cruise to cruise, the greatest concentrations of the species were always
found within the Mixed Area, and a close agreement existed between the contours
of abundance and the boundary line of the mixed water (Figs. 5, 6, and 10).
In addition to the situation in September, 1939, already described, striking cases
of conformity between the distribution of the 5. elegans population and the
extent of the Mixed Area are seen in May and June (Fig. 6). During the May
cruise stratified water was found to occupy the southern portion of the top of
the Bank extending for 30 miles or more from the southern edge toward the
center, whereas in June the mixed water largely covered this region. Corre-
sponding to this shift in the position of the water masses 5. elegans was found
to be almost completely absent from the southern half of the Bank in May, but
in June its distribution extended to the southern edge of the Bank.
In general the abundance of S. elegans tended to be relatively uniform for
all the stations within the Mixed Area during each cruise. This fact showed
that the spacing of the stations in this area was sufficiently close. The uni-
formity was no doubt due in large measure to the turbulence of the water in the
Mixed Area and would not necessarily be expected in other regions of uniform
hydrographic conditions but with less water movement.
SAGITTA ELEGANS ON GEORGES BANK 215
Sagitta elegans is therefore chiefly abundant within the Mixed Area of Georges
Bank, and during the periods covered by the present cruises, at least, this species
appears to be largely isolated from surrounding regions. Evidence has been
presented above that a small part of the population may be carried to the west
at certain seasons by the movement of water around Nantucket Shoals. It is
unfortunate that our observations could not have been extended to Cape Cod
and to the waters north of the Cape in order to ascertain whether the sagittae
of Georges Bank ever attain any important relationship with populations occur-
ring in that region. As far as our present data go, however, no significant con-
nection is indicated between the concentration of S. elegans in the vicinity of
Massachusetts Bay reported by Redfield and Beale (1940) and the population
on the Bank.
GROWTH AND BREEDING OF SAGITTA ELEGANS
Seasonal Changes in Length
The specimens of Sagitta elegans taken during the present investigation varied
greatly in size, covering a range in length from 4 mm. to 30 mm. The frequency
distribution of the sizes for all the hauls of the 1939-1940 cruises may be examined
from the histograms of Figure 7. It is seen that no specimens longer than 20 mm.
were taken in September, but individuals as long as 30 mm. occurred in January,
March, and May. The modal length increased from 16 mm. in September to
18-20 mm. in January, and to 24 mm. in March. In April the modal length
was 22 mm. These larger sizes were also represented in May and June, but in
diminishing numbers.
Specimens of Sagitta elegans as small as 6 mm. were present in the September
cruise in numbers nearly as great as the intermediate sizes. In January the
presence of a secondary mode at 8 mm. suggests the simultaneous existence of
two generations. In March and April, however, the smaller sizes were reduced
to extremely small numbers and the intermediate sizes were poorly represented.
Smaller individuals appeared in May and were more abundant in that month
than the larger categories. By early June the numerical strength of this new
crop of small Sagitta had increased ten fold and by late June they were still
abundant. The modal length increased from 6 mm. in May to 8 mm. in June.
Although in the late June cruise there were many more Sagitta in the 4 mm.
class, an even greater augmentation of the sizes larger than 10 mm. was observed.
Similar changes in the relative abundance and length distribution of this species
were encountered during the cruises of 1941.
Scrutiny of the length distributions at the various individual stations and
within the different strata revealed in general no tendencies for segregation. In
most cases both large and small specimens were represented in the same relative
proportions at the various depths at each station. Although considerable
variation in length frequency occurred from station to station, nevertheless in
most cruises there was no consistent tendency for large or small individuals to
appear in certain parts of the Bank. An exception to the foregoing statement
occurred in the cruise of May 1940, as shown in Figure 8, in which individual
frequency distributions have been plotted for each station with sufficient numbers.
In this case it is seen that at the easternmost stations only large specimens of
216
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
Sagitta elegans were taken, whereas at the western stations the smaller sizes
definitely predominated. Evidently the remrtant of the older animals persisted
chiefly in central and eastern eddies, while the production of younger individuals
was beginning most actively in the western parts of the Bank (see below).
;i
LENGTH FREQUENCY OF S. ELEGANS
1939 -'40
JUN 19-27
14
13
12
II
10
9
8
7
MAY
APR
JAN
SEPT
4 6 ' 8 10 ' 12 ' 14 ' 16 ' 18 ' 20 ' 22 ' 24 ' 26 ' 28 ' 30 '
LENGTH IN MM.
FIGURE 7. Average frequency distribution of lengths during 1939-40. Average numbers per
cubic meter of S. elegans for each cruise.
Maturity Stages and Breeding Periods
Length measurements alone are not sufficient for determining the breeding
periods of Sagitta because very great variations exist in the sizes of the three
SAGITTA ELEGANS ON GEORGES BANK
217
stages of maturity. This fact is amply demonstrated by the graphs of Figure 9,
in which the length frequency distribution of each maturity stage has been
plotted on a percentage basis for each cruise. Here it is seen that immature
specimens (Stage I) may attain a length of 16 mm. or more, but individuals as
short, as 8 mm. may definitely have attained the "intermediate" condition
(Stage II). Furthermore some specimens grew to lengths of 24 mm. or more
while still in Stage II, while other individuals became completely mature (Stage
III) at a length of 12 mm.
FIGURE 8. Length frequency distribution for individual stations in cruise of May 1940.
Length measurements of S. elegans on percentage basis for individual stations, where numbers
were sufficiently great.
From a study of the sequence of changes in the length and stage of maturity,
as presented in Figure 9, information may be derived on the seasons of growth
and reproduction of Sagitta elegans on Georges Bank. In the cruise of September
1939, the immature individuals were somewhat more numerous than either of
the other stages, but both Stages II and III were well represented. The latter
stages were much smaller in size than the corresponding groups taken during the
spring months. The modal length of the mature Sagitta was only 16 mm. in
September as compared to 23 mm. in the following March. In the January
cruise Stages I and II were encountered in about equal numbers, but very few
mature specimens were present. By March and April the majority of individuals
had matured to the Stage III condition, and remnants of these animals were
still found in diminishing numbers (and in smaller sizes) in the May and early
218
G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
MM
30
CRUISE 89 CRUISE 93 CRUISE 95 CRUISE 96 CRUISE 97 CRUISE 98 CRUISE 100
28-
0 5 K) 15 20%
STAGE I O
STAGE I ®
STAGE I •
1940
MAR
1941
CRUISE 112
1940
APR
1941
CRUISE 113
1940
MAY
1941
CRUISE 114
1940 1940
JUN
1941
CRUISE 116
FIGURE 9. Stages of maturity and length frequency distribution for all cruises. Average
values for S. elegans for all hauls in each cruise. Horizontal scale gives the percentage at each
length, subdivided into the stages of maturity as indicated by the shading. Stage I — Immature,
Stage II — Intermediate, Stage III — Mature.
June cruises. In May, however, the bulk of the catch consisted of immature
specimens, and in the June cruises Stage I was also relatively the most abundant.
Stage II was very scarce in May but appeared in increasing numbers (and in
increasing sizes) in June. It is clear that this period of relative abundance of
SAGITTA ELEGANS ON GEORGES BANK 219
immature animals corresponds to the time of great increase in the actual numbers
of smaller sizes which was noted in the previous section.
The striking difference observed in the lengths of the mature Sagitta at
various seasons of the year is correlated in a general way with temperature.
The shortest modal length for Stage III (16 mm.) occurred in September when
the temperature of the water in the Mixed Area surpassed 16° C., the highest
for the year, and the greatest modal length (23 mm.) was observed in March
when the temperature reached a minimum of about 2.5° C. (Table II). As
temperature increased through June, the mature sagittae became shorter again.
Russell (1932) reported similarly that an inverse relationship existed between the
length of the mature Sagitta elegans and the temperature of the water in the
Plymouth Area, and the same tendency was observed in the Irish Sea by Pierce
(1941). The maximum temperature recorded by Russell was about the same
as in the present investigation, but the average length of his Stage III animals
was only about 10 mm. On the other hand, the minimum temperature off
Plymouth did not fall below 8° C. and the average length of the adult Sagitta
was about 12 mm. in February, about 16 mm. in April to May 1931, and about
20 mm. in May 1930. No animals of length greater than 22 mm. were taken
by Russell, whereas specimens as long as 30 mm. were encountered on Georges
Bank. Our observations therefore agree with Russell's in revealing an inverse
relation between temperature and body length, but the actual values are quite
different. We have no information at present as to the mechanisms which
underlie these relationships.
The changes in the Sagitta population during the spring of 1941 as revealed
by the four corresponding cruises of that year, agree in general with those of the
previous spring. There is, however, consistent indication that growth and
reproduction were delayed in 1941 (Figure 9, bottom), although no important
difference in the temperature of the Mixed Area water occurred for the corre-
sponding months (Table II). In March 1941, there were relatively many more
Stage II animals and many fewer Stage III individuals than in March 1940.
The mature Sagitta dominated the scene in April 1941 and persisted in much
higher relative abundance in May of that year than had been the case in 1940.
At the same time it is to be noted that no important increase in Stage I is indi-
cated in May 1941, in marked contrast to the previous year. Nevertheless, by
early June 1941 (Cruise 116) small-sized Stage I animals appeared in very great
numbers both relatively and absolutely. Stage III was reduced to a small
remnant at this time, and there was no sign of an increase in the numerical
strength of Stage II as had occurred in the previous June. It may therefore be
concluded that in 1941 both the appearance of mature adults and the production
of the large spring crop of young took place about a month later than in 1940.
An attempt may be made from the foregoing information to ascertain the
annual cycle of growth and reproduction of Sagitta elegans on Georges Bank, but
it is obvious that the gaps in the record during months in which no data could
be obtained prevent final conclusions from being reached. It seems almost
certain, however, that the chief period of reproduction centers in April or May
because of the very high proportion of mature adults in those months, and the
appearance subsequently of very large numbers of small, immature individuals.
The adults which produced these animals apparently die off in June and the new
220 G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
crop of Sagitta matures during the summer to form a distinct generation of
adults. This supposition is strongly supported by the fact that the Stage III
animals found in September were of an entirely different size from those which
had been prevalent in the spring. The graph (Fig. 9) suggests that the spring
crop of young animals begins developing into Stage II in June with the possible
appearance of a few of the new small-sized adults. The Stage I animals present
in September may represent either the end of the spring and summer spawning
or the beginning of the reproductive activity of the new generation of small
adults. By January practically all of these small Stage III animals have disap-
peared and the immature individuals present are presumably their progeny.
It seems safe to assume that the latter then slowly mature to produce the rela-
tively large group of adults found in March and April, thus completing the cycle.
On the basis of the foregoing reasoning we may tentatively conclude that on
Georges Bank Sagitta elegans undergoes one major period of reproduction during
the spring months and that a distinct second generation is produced sometime
in the late summer or autumn. It must be borne in mind, however, that during
July and August and in the period between September and January, when no
observations were made, another complete generation could have been formed.
Russell (1932) believes that Sagitta elegans may complete a generation in as
little as 43 days during the warmer months; and he has interpreted his data from
the Plymouth Area as indicating that this species produces four or five generations
during the course of the year. Pierce (1941), however, concludes that in the
Irish Sea there is but one chief spawning period for S. elegans annually, extending
from January through May. Our present data definitely indicate the existence
of one major and one minor generation of this species in the Georges Bank area,
but do not justify as yet any assumption that further generations occurred during
the year.
DISCUSSION
Information derived from the foregoing analyses of the distribution, growth,
and reproduction of Sagitta elegans may now be examined as a contribution to
the ecology of this species on Georges Bank particularly with reference to the
currents of the region. Our previous knowledge of the occurrence of S. elegans
off the New England coast has been summarized by Redfield and Beale (1940)
in relation to their own studies of the sagittae in the Gulf of Maine. Although
no special study of Georges Bank was undertaken by these authors, occasional
stations on the Bank were occupied in the course of their survey, and at these
stations large numbers of 5. elegans were almost always encountered. These
rich hauls contrasted sharply with the situation in the central area of the Gulf
of Maine where the species was very scarce at practically every station. The
explanation offered by Redfield and Beale is that water barren in respect to
Sagitta elegans enters the Gulf each year and circulates through the central area
so rapidly that sufficient time does not exist for large populations of this species
to build up, even though the ecological conditions may be favorable in other
respects. On Georges Bank, in contrast, these authors suggest that the water
mass is sufficiently permanent to allow sagittae to accumulate and to further
augment their number through effective reproduction.
SAGITTA ELEGANS ON GEORGES BANK
221
The present investigation, based as it is on a much larger number of hauls on
Georges Bank itself, not only tends to confirm the general suggestions of Redfield
and Beale, but also provides strong evidence on the degree of permanence of the
bank water. On the other hand, scrutiny of all the present data indicates that
ecological factors other than simple transportation and accumulation play
important roles in determining the distribution and abundance of the plankton.
Redfield and Beale themselves point out that the central area of the Gulf of
Maine supports a rich endemic population of Crustacea in spite of the fact that
FIGURE 10. Comparison of the horizontal distribution of certain species of plankton in
September 1939 (cruise 89). Distribution of Sagitta elegans is compared with that of S. serrato-
dentata, S. enflata, Pseudocalanus minutus, and Calanns finmarchiciis. Values shown are average
numbers per cubic meter (per 10 cubic meters for 5. elegans) for the whole water column for all
stages of each species. Plankton Sampler hauls. Occurrence records for 5. serratodentata and
S. elegans are from the 75 cm. silk net hauls. The boundary of the Mixed Area is indicated by
the heavy broken line. The symbol "X" represents a value of less than one individual per unit
volume.
these animals are presumably subject to the same dislocating action of the
currents as was invoked to explain the scarcity of S. elegans in that area.
The agreement of the distribution of 5. elegans and of certain other species
of plankton with the disposition of the water masses on Georges Bank during the
present survey is well illustrated by the comparison for the cruise of September
1939, presented in Figure 10. Allusion has been made above to the close con-
formity of the main abundance of Sagitta elegans to the limits of the Mixed Area
222 G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
for that period. Two other species of Sagitta appeared during the September
cruise in sufficient abundance to permit significant analysis of distribution to be
made, although numbers were so small that presence or absence alone has been
indicated in the chart of their occurrence (Figure 10, lower left). It is seen that
the distribution of 5. serratodentata and 5. enflata was confined to the southern
and eastern margins of the Bank in distinct contrast to S. elegans. A remarkably
close agreement is observed between the line limiting the occurrence of the
former and the margin of the Mixed Area. S. serratodentata occurred farther up
on the Bank than did S. enflata but neither had been carried into the Mixed
Area at more than one or two points. This fact shows that a very small amount
of water, if any, was entering the Mixed Area from the south or the east at the
time of this cruise.
It is of value to compare this striking case of the separation of two morpho-
logically similar species by the hydrographical condition on the Bank with an
equally clear-cut reciprocity in the distribution of two copepods, Pseudocalanus
minutus and Calanus finmarchicus (Figure 10, upper right and lower right).
Pseudocalanus occurred at all stations on the Bank during this cruise in very
large numbers except along the southern and northeastern margins, but concen-
trations of over 1000 individuals per cubic meter were limited to the central
region and to the north central margin, this zone of abundance corresponding
closely to the Mixed Area.
In the case of Calanus, on the other hand, no specimens whatsoever were
taken in the central portion of the Bank and very low counts were obtained for
this species at every station within the Mixed Area (Fig. 10). Similarly small
catches of Calanus were made along the extreme southern margin of the Bank
and to the northeast, but considerable numbers were taken at the stations to
the north and west toward the Gulf of Maine, and in the zone between the
Mixed Area and the southern margin of the Bank. The tongue of water, rich
in Calanus, which appears extending across the eastern end of the Bank and
curving south and then west, is a clear indication that at the time of this cruise
a current carrying Calanus from the Gulf of Maine was flowing around the margin
of the Mixed Area and forming a wedge between it and the water masses to the
south. At the same time mixed water, barren in respect to Calanus, from the
central eddy of the Mixed Area appears to have been draining off to the west
down the middle of the Bank. Calanus finmarchicus therefore appears to be a
species which can endure neither the homogeneous water of the Mixed Area
nor the warm, saline conditions to the south, but which thrives in the water of
the Gulf of Maine. Thus Calanus is similar to S. serratodentata and S. enflata
in being sharply excluded from the Mixed Area of Georges Bank, whereas Pseudo-
calanus and S. elegans are chiefly abundant within it.
Although one could argue that even if S. serratodentata and S. enflata found
their way on to Georges Bank, they could not survive there because of lower
temperatures and salinities, it is impossible to invoke these factors as preventing
the occurrence of Calanus finmarchicus in the central part of the Bank. As we
have seen, the water of the Mixed Area is chiefly derived from the Gulf of Maine
where Calanus is abundant, and the temperature and salinity values of this
water are generally intermediate between those of the upper and lower strata
of the Gulf. Some other factor must be found which could prevent the repro-
SAGITTA ELEGANS ON GEORGES BANK
duction of Calanus in the Mixed Area or which could cause its destruction (or
both), and which does not affect Pseudocalanus adversely. Perhaps sediment
resulting from the turbulence in the Mixed Area is harmful to Calanus or possibly
some essential element is lacking, such as accessible bottom water of low temper-
ature (cf. Bigelow and Sears, 1939), or a necessary food organism. On the other
hand, destruction of Calanus might be brought about chiefly by a predator, and
as such S. elegans suggests itself for future study since it populates so abundantly
the very area from which Calanus is removed.
Since other ecological influences besides the purely mechanical action of the
current system are apparently controlling the occurrence of Calanus on Georges
Bank, it seems probable that additional, as yet undetermined, factors may be
important in accounting for the special richness of the population of Sagitta
elegans in the Mixed Area. It now appears well established that the relative
permanency of the bank waters makes possible the maintenance of an adequate
breeding stock of S. elegans from one period of reproduction to another, but, in
addition the data presented above indicate that the breeding and growth of this
species is especially successful in the mixed bank water during the spring months,
and perhaps at other seasons as well.
This suggestion that the great abundance of Sagitta elegans within the Mixed
Area of Georges Bank is due not merely to mechanical accumulation but also to
especially favorable local conditions, is supported by observations from other
regions. Bigelow and Sears (1939) consider that this species is regularly endemic
in the waters overlying the inner half of the continental shelf from Cape Cod
south possibly to the offing of Chesapeake Bay. There are no large, permanent
eddies in this area. Russell (1939) pointed out that S. elegans occurred in
dominant numbers around the British Isles in areas where mixed oceanic and
coastal water occurred. This author does not report any accumulation of the
species in these areas through a mechanical action of the currents, but states
that "The incursion of oceanic water gives rise to conditions in which a different
plankton community, typified by Sagitta elegans, flourishes."
It is obviously of importance to inquire what characteristic of the mixed
water of Georges Bank may make it an especially favorable environment for the
reproduction and growth of Sagitta elegans. Temperature and salinity may
apparently be ruled out since we have seen that the values for these factors are
within the range of those found in the central region of the Gulf of Maine where
this species is relatively scarce. Three other possibilities suggest themselves:
(1) the turbulence and the attendant vertical homogeneity of the water, (2) the
shallowness of the water, and (3) the existence of some element in the water
originally derived from the shore or from the bottom. Although we do not
have the means at present to decide between these, or other possibilities for
Georges Bank, some evidence may be obtained from the ecological conditions of
other regions of abundance for this species. Such comparison appears to elimi-
nate vertical mixing of the water, per se, as particularly favorable since this
condition does not characterize most of the other areas of occurrence of S. elegans.
The waters of the continental shelf to the south, and of Massachusetts Bay to
the north, are both definitely stratified during the warmer portion of the year.
Furthermore, breeding was found to be unsuccessful in the Bay of Fundy (Hunts-
man and Reid, 1921) where vertical mixing is strong.
224 G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
The shallowness of the water might be regarded as a beneficial condition for
5. elegans since all observations agree that this species occurs chiefly in the shoal
water of the continental shelf or of off-shore banks both along the North American
coast and around the British Isles. It is doubted, however, whether the nearness
of the bottom could be a direct, favorable influence (a) because there is no evi-
dence that this species has any ecological dependence on the bottom, (b) because
its distribution on Georges Bank showed no relation to the depth contours, and
(c) because in other regions it has been found to be as abundant in water 100 m.
to 200 m. deep as in shallower zones.
The third suggestion, that some beneficial derivative of the shore or the
bottom occurs in the shallower water of the coastal areas or the banks, remains
as a possible, though vague explanation. Eraser (1939) remarks with reference
to Sagitta that plankton in general can withstand "fairly big" physical changes
and hence the very distinct separation in the distribution of species means some
biological change in the water masses. The very definite tendency for abundant
populations of S. elegans to occur in shoal areas therefore suggests the presence
in the water of some chemical element derived from the shore or bottom or some
food organism dependent on the bottom, which does not exist in the water of
deeper regions.
We conclude, therefore, that Sagitta elegans is chiefly abundant within the
Mixed Area of Georges Bank, first, because the relative permanence of this
water mass allows it to accumulate there; second, because water of harmfully
high temperature and salinity from the south is excluded; and third, because
some indirect influence from the shore or bottom, absent in the deeper water
to the north, favors its reproduction and growth. It remains for the future to
re-examine the water of the Mixed Area in order to ascertain what conditions
of feeding or other circumstance, render this water particularly favorable for
S. elegans and certain other types of plankton, and particularly unfavorable for
other, closely related species.
Finally, it may be emphasized that the observations from the eleven cruises
of this investigation have shown that Georges Bank supports a relatively abun-
dant population of S. elegans throughout the year, and that the center of concen-
tration of Sagitta was always found within the Mixed Area. Furthermore, the
contours of the Sagitta population were shown to conform in general to the
limits of the Mixed Area. Frequently this rich area was completely surrounded
by water in which S. elegans was very scarce or absent. It appears then that
this species is an adequate indicator for the presence of Mixed Area water. We
know, as a result, that the eddy of homogeneous water on Georges Bank may be
regarded as permanent to the extent to which the population of Sagitta elegans
has been shown to maintain its integrity there from season to season. It is of
special interest to note that a nucleus of this species apparently retained its
position on the Bank throughout the winter, during the period when the break-
down of stratification in the surrounding areas might be expected to make
possible a flow of Gulf water directly across the Bank. The bubble of mixed
water on the Bank therefore either fails to be dislodged by hydrographic forces,
or is renewed so slowly that the population of Sagitta is able to maintain itself
within the Mixed Area despite the water movement. By similar application of
SAGITTA ELEGANS ON GEORGES BANK 225
these findings, it should be feasible to employ 5. elegans as an indicator to trace
the movements of Mixed Area water in future studies of the ecology of the region.
SUMMARY
1. The quantitative distribution, size, and stages of maturity of Sagitta elegans
in the waters of Georges Bank have been determined from plankton hauls made
on a network of stations occupied during 11 cruises from September 1939, to
June 1941.
2. The area of relatively homogeneous water overlying the central portion of
Georges Bank was found to change in extent from cruise to cruise, but to be
sharply delineated from the surrounding stratified water masses, and has been
designated as the "Mixed Area."
3. The abundance of 5. elegans varied in individual hauls from a maximum
of 165 specimens per cubic meter to zero, but averaged more than 10/m.3 at
stations within the Mixed Area. The deeper hauls were numerically richer than
the shallow hauls at more than half the stations. The existence of a diurnal
vertical migration was revealed in certain cases, but varied greatly from cruise
to cruise.
4. In horizontal distribution the greatest concentrations of S. elegans were
found within the central homogeneous water mass of the Bank and a close
agreement was disclosed between the contours of abundance and the boundary
line of the Mixed Area. A center of abundance for this species existed on the
Bank throughout the year.
5. The modal length of the mature sagittae increased from 16 mm. in Septem-
ber, when the water temperature was the highest, to 23 mm. in March, when
the temperature was at the minimum for the year. Some specimens as long as
30 mm. were encountered.
6. The chief period of reproduction for 5. elegans on Georges Bank centered
in April in 1940 and in May in 1941. Following these periods very large numbers
of small, immature individuals appeared. Evidence is presented that a distinct,
second generation was produced during the late summer or autumn.
7. S. serratodentata and 5. enflata, which were taken chiefly in the September
cruise, occurred entirely outside the margin of the Mixed Area. Comparison is
made with a similar case of reciprocal distribution found for. two species of
copepods: Calanus finmarchicus, which was excluded from the Mixed Area, and
Pseudocalanus minutus, which was chiefly abundant within the Area.
8. Our observations support the suggestion of Redfield and Beale that S.
elegans tends to accumulate on Georges Bank because of the relative absence of
dislocating currents. However, reasons are advanced for believing that other
characteristics of the Mixed Area water should be scrutinized as being particularly
favorable for the growth and reproduction of S. elegans, and particularly unfavor-
able for the existence of other species of plankton.
9. The persistence of the population of 5. elegans on Georges Bank throughout
the year is a valuable index of the degree of permanence of the homogeneous
bank water. The species recommends itself as an indicator for tracing move-
ments of the Mixed Area water in future studies.
226 G. L. CLARKE, E. L. PIERCE, AND D. F. BUMPUS
LITERATURE CITED
BIGELOW, H. B., AND M. SEARS, 1939. Studies of the waters of the continental shelf, Cape Cod
to Chesapeake Bay. III. A volumetric study of the zooplankton. Memoirs of the Mus.
Comp. Zool. at Harvard College, 54: 189-373.
CLARKE, G. L., 1934. Factors affecting the vertical distribution of copepods. Ecological
Monographs, 4: 530-540.
CLARKE, G. L., AND D. F. BUMPUS, 1940. The Plankton Sampler — an instrument for quantitative
plankton investigations. Linnological Society of America, Special Pub., (No. 5): 1-8.
FRASER, J. H., 1939. The distribution of Chaetognatha in Scottish waters in 1937. Jour, du
Conseil, 14: 25-34.
HUNTSMAN, A. G., AND M. E. REID, 1921. The success of reproduction in Sagitta elegans in the
Bay of Fundy and the Gulf of St. Lawrence. Trans. Roy. Canadian Inst., 13: 99-112.
ISELIN, C. O'D., 1939. Some physical factors which may influence the productivity of New
England's coastal waters. Sears Found. Jour. Afar. Res., 2: 74-85.
PIERCE, E. L., 1941. The occurrence and breeding of Sagitta elegans Verrill and Sagitta setosa
J. Miiller in parts of the Irish Sea. Jour. Marine Biol. Assoc., 25: 113-124.
REDFIELD, A. C., AND ALICE BEALE, 1940. Factors determining the distribution of populations
of Chaetognaths in the Gulf of Maine. Biol. Bull., 79: 459-487.
RILEY, GORDON A., 1941. Plankton studies. IV. Georges Bank. Bull. Bingham Oceanographic
Coll., VII: Art. 4, 1-73.
RILEY, GORDON A., 1942. The relationship of vertical turbulence and spring diatom flowerings.
Sears Found. Jour. Mar. Res., 5: 67-87.
SEARS, MARY, 1941. Notes on the phytoplankton on Georges Bank in 1940. Sears Found. Jour.
Mar. Res., 4: 247-257.
RUSSELL, F. S., 1932. On the biology of Sagitta. The breeding and growth of Sagitta elegans
Verrill in the Plymouth area, 1930-31. Jour. Mar. Biol. Assoc., 18: 131-146.
RUSSELL, F. S., 1933. On the biology of Sagitta. IV. Observations on the natural history of
Sagitta elegans Verrill and Sagitta setosa J. Miiller in the Plymouth area. Jo.ur. Mar.
Biol. Assoc., 18: 559-574.
RUSSELL, F. S., 1939. Hydrographical and biological conditions in the North Sea as indicated
by plankton organisms. Jour, du Conseil Intern, pour VExpl. de la Mer, 14: 171-192.
THE MORPHOLOGY AND LIFE HISTORY OF THE DIGENETIC
TREMATODE, ZOOGONOIDES LAEVIS LINTON, 1940
HORACE W. STUNKARD
(New York University}
Linton (1940) described the adult stage of Zoogonoides laevis and distinguished
between this species and Z. viviparus (Olsson, 1868) Odhner, 1902, the type and
only other known representative of the genus. He found the worms in the
intestine of Tautoga onitis and an immature specimen from the round herring,
"Etrumens sadina" (= Etrumeus teres) was referred provisionally to Z. laevis.
During the summer of 1942, tailless cercariae were found emerging from
Columbella ( — Mitrella Rizzo) lunata collected in the Woods Hole region. Their
striking resemblance to the cercariae of Zoogonus lasius (Leidy, 1891) Stunkard,
1940 indicated that the two were closely related. Furthermore, their morpho-
logical agreement with Zoogonoides laevis suggested that they might be larvae of
the latter species. Experiments demonstrated the correctness of the hypothesis
and the successive stages in the life cycle have been obtained. The cercariae
develop in sporocysts in the lymph spaces of C. lunata, penetrate into Nereis
virens where they become metacercariae, and sexual maturity is attained in the
intestine of T. onitis. The eggs are large, without shells, and contain active,
ciliated miracidia when extruded. The larvae hatch in sea water and invade the
snails where the asexual generations are produced. The life history was reported
in abstract (Stunkard, 1942).
EXPERIMENTS
The methods employed in the present study were similar to those described
by Stunkard (1938, 1941) in reports on the life history of Zoogonus. Over 2000
specimens of Columbella lunata were collected from algae taken at several loca-
tions. They were isolated in groups of ten or more in large stender dishes and
observed morning and evening for five days to obtain preliminary information
concerning the incidence of infection and also to obtain infective cercariae for
life history experiments. Water in the dishes was changed twice daily and bits
of algae were provided as food for the snails. When cercariae appeared in a
dish, the snails were isolated individually to obtain the one or more which harbored
the parasite. Of the snails from which no larvae emerged, several hundred
were crushed and examined to secure more complete information concerning the
total incidence of infection. In different collections the total infection varied
from 0.5 to 5 per cent, although about one-fourth of the infected snails did not
liberate cercariae.- The snails are small and the cercariae are relatively large;
consequently the number of cercariae which emerged from any snail was small,
usually two to five in 24 hours. Sometimes an infected snail would not liberate
cercariae for three or four days. The larvae are hardy and may live for four
days in sea water. In form and behavior, they resemble those of Zoogonus lasius.
227
228 HORACE W. STUNKARD
They emerge during the day and at night. The posterior end of the body exudes
a sticky substance and bits of debris adhere to it. The secretion causes the
cercariae to stick to the wall of a pipette and it is difficult to transfer them from
one dish to another. They adhere to the substratum or move about by alternate
attachment of the ends of the body, using the oral sucker to attach the anterior
end. Ordinarily the larvae do not encyst in fresh sea water, but occasionally
one would extrude cystogenous material on a slide if the water evaporated enough
to seriously disturb the osmotic equilibrium. The cystogenous secretion is often
emitted when the larvae are placed in solutions of vital dyes, and sometimes
encystment is complete. If sporocysts containing fully grown cercariae are
removed from a snail and left for several hours in sea water, and especially if
the sporocysts become moribund, the older cercariae may encyst in them. Also,
sporocysts removed from dead snails may contain encysted cercariae.
Infection of Second Intermediate and Final Hosts
Since the cercariae are unable to swim, and since they do not normally encyst
in sea water, it was apparent that the next host must be a bottom-dwelling
animal. Accordingly, various molluscs, crustaceans and worms were placed in
finger bowls with normally emerged larvae. The larvae disappeared in a few
hours from the dish with Nereis virens, whereas they persisted for at least two
days in dishes with the other animals tested. A parapodium was cut from a
living specimen of Nereis and placed in a stender dish with four cercariae; four
hours later, three of them had penetrated into the parapodium and two had
encysted. These observations indicated that Nereis may serve as an intermediate
host, although they do not preclude the possibility that other animals also may
be utilized in nature. When it was observed that the larvae would encyst in
Nereis, mass infection was attempted. Sixteen infected C. lunata and four
Nereis were placed in a small aquarium provided with about 2 cm. of sand on
the bottom and with a cheesecloth cover to permit the entrance of sea water and
to prevent the escape of the snails and worms. These worms were exposed to
cercariae for two weeks and then fed to tautogs which had been isolated without
food for seven weeks. The fishes were dissected three days later and dozens of
young Z. laevis, some of them hardly larger than cercariae, were recovered. In
addition, there were other, larger and more mature specimens of Z. laevis which
undoubtedly represented a natural infection, acquired by the fishes before they
were caught. Tautogs which were dissected immediately after capture were
always infected; the number of worms taken from the intestine varied from a
few to about three hundred, and in heavy infections all stages of development
from recently excysted metacercariae to gravid specimens were found. The
small worms of natural infection were morphologically indistinguishable from
those of the experimental infection. It is apparent from these experiments that
the cercariae from C. lunata encysted in Nereis, that they later became established
in the intestine of the tautog, and that they are larvae of Z. laevis. Furthermore,
it is apparent that the metacercariae are infective for the final host soon after
encystment.
Infection of the First Intermediate Host
Gravid worms from natural infections often contain many active, ciliated
miracidia, enclosed in membranous capsules. Since the worms have small,
MORPHOLOGY AND LIFE HISTORY OF Z. LAEVIS
deficient vitellaria, the eggs lack shells. When such gravid specimens were
compressed under a coverglass for study or for fixation, occasionally the egg
nearest the genital pore was expelled. In a few instances, after the addition of
tap water, the membrane ruptured and the larva emerged and swam about.
Dilution of the sea water is not necessary, however, to induce hatching of the
miracidia. When mature worms are removed from the fish to sea water, they
usually extrude eggs and the miracidia emerge in a few hours. The larvae
elongate in swimming, rotate on the long axis, and progress rapidly in a spiral
path. They become uniformly distributed in the water and no obvious reaction
to light was observed. Since the miracidia emerge from the eggs and swim
vigorously, it is apparent that they penetrate the snail host to establish the
infection. Normally emerged, free-swimming miracidia were not noticeably
attracted toward specimens of C. lunata placed in a dish with them. It appears
that their contact with snails is accidental and larvae would sometimes make
contact with a snail and then swim away. Penetration by the larvae was not
observed and it seems probable that they enter the branchial cavity of the snail
with water and then invade the tissue. This hypothesis is supported by the
location of primary sporocysts dissected from experimentally infected snails.
To obtain experimental infection of C. lunata, 50 snails, which had been
isolated for two wreeks without the appearance of any cercariae, were placed in a
small tank with two tautogs and left there for 19 days. At the end of that time
30 of them were recovered and transferred to finger bowls. The fishes were
dissected and more than two hundred mature specimens of Z. laevis were taken
from them. Five of the snails which had been exposed to infection were dissected
and young sporocysts were found in two of them. The parasites, undoubtedly
primary sporocysts, were small, oval to irregularly shaped sacs containing groups
of germinal cells. The other snails were kept in a finger bowl and those that
died or became weak were crushed and examined. Sporocysts which contained
unidentifiable germ masses were observed in the snail tissues and at the end of
the season, seven weeks after the snails had been placed in the tank with the
fishes, the twelve remaining snails were killed and examined. Ten of them
contained sporocysts; six were heavily and four were lightly infected. Each of
the heavily infected snails contained hundreds of sporocysts; each of the lightly
infected ones contained fifty to one hundred sporocysts. The number of sporo-
cysts suggests that they were the second or daughter generation. Several of the
sporocysts contained large, elongate germ balls but differentiation had not
proceeded to a point where they could be recognized as either young sporocysts
or cercariae. The snails had been examined daily for emerged cercariae and
since none were observed, it is evident that the infection of the snails was an
experimental one. The cercariae are produced in sporocysts, but the number of
sporocyst generations and the time from entrance of miracidium to emergence of
cercariae is yet unknown. Due to the slow development of the parasites and
the shortness of the working season, it was impossible to complete the cycle in
experimentally infected hosts, but experimental infection of the definitive and
both intermediate hosts was secured and, accordingly, the data appear to provide
convincing proof of the life cycle.
Both adults and larvae of Z. laevis were studied alive, with and without
vital staining and after fixation and staining. Specimens were fixed in the
230 HORACE W. STUNKARD
extended condition under coverglass pressure for whole mounts and others
without pressure for sections, in order that distortion from compression could be
recognized.
DESCRIPTIONS
The description of the sexually mature stage of Z. laevis given by Linton
(1940), although limited to gross morphology, is adequate for identification of
the species. Since the specimens of the present study agree substantially with
the account of Linton and are from the same host and the same locality, their
identity with those of Linton is assumed.
The Adult
The worms are pale yellow to reddish in color and the larger ones are con-
spicuous on the opened intestine of the fish. They occur throughout the length
of the intestine, although the majority are located in the posterior portion.
Often they are deep between the villi and they may adhere tenaciously, especially
with the powerful acetabular sucker. On removal, they frequently pull away
the portion of the intestine grasped in the acetabulum (Fig. 1). In sea water
they are relatively sluggish and tend to remain in a contracted condition. The
body is very muscular and the shape varies with the degree of extension. Typi-
cally the worms are terete, fusiform, with the posterior half of gravid specimens
saccate and distended by many uterine coils. The preacetabular region is more
active and when extended it tends to bend ventrally forming a shallow ventral
concavity.
Gravid specimens (Fig. 2) fixed under a coverglass, stained and mounted,
vary from 0.6 to 1.1 mm. in length and 0.2 to 0.45 mm. in width. For well
extended specimens, it is necessary to compress the worms, which increases their
width. The acetabulum is slightly anterior to the middle of the body and
measures from 0.195 to 0.27 mm. in diameter. As noted by Linton, its aperture
is transverse, but he made no mention of the peculiar muscular development
which determines the shape. The opening of the sucker is provided with a
powerful sphincter which, when contracted, forms thickened muscular masses
(Figs. 1, 2) anterior and posterior to the aperture. These masses may protrude
EXPLANATION OF PLATE
ABBREVIATIONS
am anterior germinal mass ov ovary
cs cirrus sac pd penetration gland ducts
ed excretory duct pg penetration gland
em embryo pm posterior germinal mass
ev excretory vesicle sr seminal receptacle
gd gland duct ts testis
gp genital pore ut uterus
in intestine vg vitelline gland
All figures are of Z. laevis
FIGURE 1. Sagittal section through the acetabulum of adult, showing the sphincter and
lumen filled with tissue from intestine of the host; anterior end at left of the figure.
FIGURE 2. Adult, whole mount, flattened under coverglass, stained and mounted; dorsal
view.
FIGURE 3. Sporocyst from Columbella lunata, natural infection, with developing cercariae.
FIGURE 4. Cercaria, composite drawing from free-hand sketches of living larvae, showing
details of structure.
MORPHOLOGY AND LIFE HISTORY OF Z. LAEVIS
231
!-— ut
cs-
2
>a — IP)
pm-
PLATE I
232 HORACE W. STUNKARD
into the lumen, giving the opening of the sucker a dumb-bell shape. The cuticula
is thick and in living specimens shows reticulate furrows. Minute, straight,
retrose spines are embedded in it. They measure about 0.006 mm. in length;
their bases are adjacent to the basement membrane and their tips barely protrude
from the surface of the cuticula. The spines are seldom visible on stained and
cleared specimens but show well in sections stained with iron haematoxylin.
They are somewhat smaller in the area around the mouth and become sparse on
the posterior part of the body. There are small papillae at the anterior end;
presumably these are the structures which in the cercaria bear sensory bristles.
The oral sucker measures from 0.1 to 0.145 mm. in diameter, with the mouth
subterminal in position. There is a short prepharynx and the commissure of
the nervous system, although morphologically prepharyngeal, usually appears to
lie above the pharynx. The pharynx measures 0.03 to 0.06 mm. in diameter
and the esophagus extends posteriad about one-half the distance to the acetabulum
where it joins the intestinal ceca. The ceca pass backward on the dorsal side
of the body above the cirrus sac, metraterm and testes, and end just behind
the level of the seminal receptacle. They have a narrow and almost uniform
diameter.
The excretory system of the adult is morphologically identical, except for
slight changes incident to growth and sexual maturity, with that of the cercaria
(Fig. 4). The pore is terminal and a short canal leads from it to the bladder.
The cuticula is thickened at the posterior end of the body and the circular muscles
are modified to form sphincters at the outer and inner ends of the excretory
canal. When the pore is closed the wall of the canal may lie in .longitudinal
folds which cause the lumen to be radiate in cross section. At the posterior end
of the worm the longitudinal muscles of the body wall converge in bands and are
inturned and inserted on the wall of the excretory canal. From this region
other muscles continue meridionally in the wall of the excretory bladder, giving
the rosette appearance depicted in the figure by Linton. It is the contraction
of these muscles which expels the contents of the bladder. The bladder is
saccate and when filled may extend one-third of the distance to the acetabulum.
From the antero-lateral surface on each side, a common primary collecting duct
passes forward in a sinuous course to the level of the acetabulum. Here it
divides into anterior and posterior secondary branches. The anterior duct
divides near the bifurcation of the alimentary tract into two tertiary ducts.
One passes forward and at the level of the pharynx divides into two capillaries
which lead to the anterior pair of flame cells; the other turns backward and
divides into two capillaries that lead to a pair of preacetabular flame cells. The
posterior secondary collecting duct divides in a pattern which is the counterpart
of the anterior one. The flame cell formula therefore is 2[(2 + 2) + (2 + 2)].
The location of the flame cells and tubules of the cercaria is shown in Figure 4
and the system persists with only minor changes, due to differential growth,
in the adult condition. The wall of the excretory bladder contains circular as
well as longitudinal fibers and the cells of the epithelial lining bear cilia or striated
brush borders. The openings of the collecting ducts are surrounded by areas
where the ciliary beat is conspicuous. The walls of the collecting ducts contain
circular and longitudinal fibers and the lumen is ciliated; the ciliary beat is
toward the bladder. Ciliated patches occur in other portions of the collecting
MORPHOLOGY AND LIFE HISTORY OF Z. LAEVIS
system and the ciliated tufts of the flame cells measure 0.009 to 0.12 mm. in
length.
The testes are ventral, situated at the sides of the acetabulum, although
they may be displaced forward or backward until they lie anterior or posterior
to the sucker. They are oval, compressed laterally and in gravid specimens
measure from 0.12 to 0.17 mm. in length and 0.07 to 0.12 mm. in width. Vasa
efferentia arise from the dorso-antero-median faces of the testes and unite to
form a single duct which immediately enters the posterior end of the cirrus sac
where it expands to form a bipartite, sigmoid seminal vesicle. The duct leading
from the vesicle is surrounded by cells of the prostate and usually contains large
secretory droplets. The cirrus is spined and although it was not observed in the
extruded condition, the structure of the parts indicates that it is eversible. The
cirrus sac is large and extends from the level of the acetabulum to the common
genital pore located near the right ventral margin at the level of the bifurcation
of the alimentary tract. The posterior end of the sac is dorsal in position and
it curves anteriad, laterad and ventrad to the pore. The opening of the cirrus
sac is below and slightly behind that of the metraterm.
The ovary is dorsal, immediately posterior to the acetabulum, either median
or lateral, right or left. It is spherical to oval and in gravid specimens measures
from 0.085 by 0.076 to 0.12 by 0.11 mm. The oviduct arises at the ventral,
posterior margin of the ovary and turns dorsally under the anterior end of the
seminal receptacle. Here it receives a very short duct from the receptacle.
From the posterior side of this short duct, Laurer's canal passes posteriad and
then dorsad by a sinuous course to open at the surface. The oviduct, after
receiving the duct from the seminal receptacle, turns ventrad and toward the
left where it expands and receives a short duct from the vitelline gland. The
female duct then continues ventrad and posteriad forming the initial portion of
the uterus. The uterus coils about and when filled with embryos, occupies the
posterior half of the body. Its course becomes irregular and impossible to follow.
The seminal receptacle is dorsal in position, immediately behind the ovary, with
the anterior end of the vesicle above and partly overlapping the posterior end of
the ovary. The vitelline gland is ventral, below and often slightly lateral to the
seminal vesicle. Usually the anterior end of the vitelline gland is slightly
anterior to the anterior end of the seminal vesicle. In the specimen shown in
Figure 2, the structures are somewhat distorted by pressure exerted in flattening
the worm. There is no "shell gland" and the miracidia develop in thin-walled,
membranous sacs. The terminal portion of the uterus has strong muscle walls.
It passes anteriorly below the ovary, turns dorsally at the right side of the
cephalic portion of the ovary and continues across the right dorsal side of the
acetabulum, passes below the cecum of the right side and turns ventrad above
the terminal portion of the cirrus sac to open at the common genital pore. The
development of the larvae in the uterus is similar to that in Zoogonus lasius.
•
The Miracidium
The uterus of Z. laevis is filled with developing miracidia and the terminal
coils contain ciliated larvae. The fully formed miracidium is oval, pointed
anteriorly and entirely covered with long, closely-set, powerful cilia. In the egg
membrane, the cilia beat vigorously and the larva performs muscular movements;
234 HORACE W. STUNKARD
the anterior tip is frequently protruded, turned about and then retracted. The
anterior portion of the larva contains two gland cells with ducts which open at
the tip; droplets were observed emerging from these openings. The nuclei in
the anterior portion of the miracidium were vesicular and stained faintly; in the
posterior portion there is a group of nuclei which stained deeply and which
probably are those of germinal cells. There are two flame cells, one anterior
and the other posterior in position, but the excretory ducts could not be followed.
The number and arrangement of the ciliated epithelial cells were not determined ;
their nuclei are flattened and irregular in shape. The egg membrane is flexible
and the shape varies with pressure; eggs in sea water and without a coverglass
measured 0.076 to 0.08 mm. in length and 0.028 to 0.032 mm. in width. The
miracidium is about 0.065 mm. long and 0.028 mm. wide and when the egg
emerges from the worm into sea water the beat of the cilia is noticeably increased.
Sporocyst Generations
The miracidia penetrate into C. lunata and produce sporocysts but, as noted
previously, the number of sporocyst generations in the life cycle of Z. laevis was
not determined. Snails infected in the laboratory during the summer did not
produce cercariae, but from these snails, exposed to miracidia for nineteen days
and dissected six weeks later, large numbers of young sporocysts were recovered.
When fixed and stained, they varied in size from 0.05 by 0.04 mm. to 0.144 by
0.11 mm., and most of them contained groups of germinal cells and germ balls,
so immature and undifferentiated that it was quite impossible to determine
whether they would become daughter sporocysts or cercariae. The young
sporocysts were much smaller than cercariae. The smaller sporocysts were very
numerous; in snails with a smaller number there was a corresponding increase in
size. Whether all of these sporocysts belonged to a first daughter generation
could not be determined. It is possible that some of the larger ones were first
generation, i.e., primary sporocysts, and that some of the smallest ones were
third generation. One of the larger ones contained five daughter sporocysts and
15 to 20 germ balls of varying sizes. Another, in addition to germ balls, con-
tained two embryos that were as large as daughter sporocysts but were undiffer-
entiated; they resembled developing cercariae but could not be identified posi-
tively. The sporocysts occupy the haemocoele of the snail.
In naturally infected snails, all of the sporocysts contained germ masses and
developing cercariae; no sporocysts containing identifiable daughter sporocysts
were observed. These sporocysts (Fig. 3) were oval to elongate, colorless sacs,
with a birth pore at one end. When fixed and stained, they varied in size from
0.25 by 0.2 mm. to 0.86 by 0.32 mm. and usually contained several young cercariae
in addition to masses of germinal tissue in various stages of development. All
of the sporocysts were motile and the non-gravid ones were very active, elongating
and shortening, bending and twisting. They changed from an oval to a cy-
lindrical shape in which the length was as much as eight times the diameter.
When elongate they would often bend in a C-shape and then thicken at one end,
becoming clavate in form. In locomotion the anterior end is protruded as a
long, slender process. It then begins to thicken near the tip until a bulbous
enlargement is formed at or near the end. This enlargement increases in size as
the contraction of circular and longitudinal muscles in the more posterior portion
MORPHOLOGY AND LIFE HISTORY OF Z. LAEVIS
thrusts the body forward, leaving an attenuated, tail-like posterior end. This
region is then pulled forward and the cycle of events is repeated. Apparently
either end' may precede in locomotion.
The Cercaria
The cercariae emerge from the sporocysts before they are entirely mature
and complete their development in the lymph spaces of the snail. Most snails
with old infections have cercariae free in the haemocoele. Normally emerged
cercariae (Fig. 4) measure 0.2 to 0.5 mm. in length and 0.06 to 0.16 mm. in
width. The acetabulum is 0.07 to 0.08 mm. in diameter. The acetabular
sphincter is developed in the cercaria and the aperture of the sucker is transverse.
The cuticula is spined. About the anterior end of the larva there are small
papillae, each of which bears a fine bristle. The oral sucker is 0.06 to 0.065 mm.
in diameter. The preoral region bears a simple, pointed stylet, 0.018 to 0.02 mm.
long, which is directed anteriad. The mouth is subterminal; there is a short
prepharynx and the pharynx measures 0.02 to 0.027 mm. in diameter. Its
lumen is diagonal, from anterodorsal to postero ventral and in extended specimens
the pharynx is usually longer than wide. The esophagus extends about half way
to the acetabulum where it joins the digestive ceca which pass laterad and
posteriad, ending blindly near the level of the anterior margin of the excretory
bladder. In living specimens stained with neutral red the ceca are easily traced
since they have a deep red color. The excretory system is fully developed in
the cercarial stage. The location of the flame cells and ducts is shown on the
right side of Figure 4. The bladder is lined with large epithelial cells and often
contains refractive concretions. The reproductive organs are represented by
two cellular masses (Fig. 4), one in front and the other behind the acetabulum.
The anterior cells form the copulatory organs; the posterior cells give rise to the
gonads, the female accessory structures and the uterus.
The unicellular glands of the cercaria are numerous and exceedingly hard to
differentiate. The staining of living specimens with vital dyes and of fixed
specimens with various cytoplasmic stains has not provided sufficiently clear
distinctions for certain identification. The cells of any given type do not stain
uniformly and in the penetration glands especially, the secretion may not stain
at all in the cell body but stains more intensely as it passes along the duct.
The age and condition of the cellular inclusions are apparently variable and the
staining reaction varies accordingly. There are many cystogenous glands
scattered over the body and they open to the surface both dorsally and ventrally.
Near the posterior end of the body there are a number of glandular cells whose
ducts pass posteriad and some of them open at or near the excretory pore. It is
probable that these cells secrete the sticky material by which the larvae are
attached. Anteriorly there are two clusters of glandular cells on each side of
the body. There are about eight pairs of penetration glands, situated lateral
and anterior to the acetabulum. Their ducts pass forward on each side of the
body and behind the oral sucker may occasionally separate into three bundles,
one of which passes mediad to the others which lie on the lateral side of the sucker.
All open to the surface beside and below the stylet. The ducts are twisted about
each other and their number could not be determined with certainty. Anterior
and lateral to the penetration glands and partially overlapping them, there are
236 HORACE W. STUNKARD
other glands, probably six on each side, whose ducts pass forward and mediad,
below the ducts of the penetration glands, and open into the prepharynx. The
function of these glands is quite unknown.
The Metacercaria
Metacercariae were recovered from the parapodia and body wall of Nereis
virens at intervals from one day to one month after the polychaetes were exposed
to the cercariae. Each larva was enclosed in a thin, non-cellular capsule, pro-
duced by the cystogenous material of the cercaria. The body was bent ventrally,
its dorsal surface applied to the cyst wall, with the two ends adjacent or over-
lapping. The parasites induced proliferation of fibroblasts in the tissues of the
host and became enclosed in connective tissue capsules. When the host tissue
was teased apart in sea water, the cysts fell out. The cysts were spherical to
oval and immediately after encystment measured from 0.12 to 0.18 mm. in
diameter. If the cysts were in locations where they were not subjected to
pressure they remained spherical, if they were between muscle layers and com-
pressed, they became oval. At the end of two weeks the cysts were noticeably
larger, the larvae had grown, the excretory vesicles were filled with concretions,
the gland cells were reduced but still recognizable, and the stylets were somewhat
smaller. With the gradual resorption of larval structures, there was a corre-
sponding development of the reproductive organs. These structures, represented
in the cercaria by two groups of deeply staining cells, had begun to assume
definitive form, although the gonads of metacercariae removed from Nereis four
weeks after exposure to infection, were no further developed than those of the
cercariae of Z. lasius.
DISCUSSION
Life history studies require precise and accurate determination of the species
of animals used in the investigation. In describing the worms from the tautog
as a new species, Zoogonoides laevis, Linton compared them with the descriptions
of Z. viviparus (Olsson, 1868) Odhner, 1902 as given by Olsson (1868), Odhner
(1902) and Nicoll (1907). These accounts are at variance in certain respects.
Odhner regarded Olsson's description as incorrect in certain particulars and
Nicoll's description agrees in the main with that of Odhner. Although Odhner's
measurements of the worm and of the oral sucker agree with those of Olsson,
Odhner found the acetabulum twice as large as the oral sucker, an observation
not in agreement with the findings of Nicoll. Olsson reported Z. viviparus as
rare, he did not find it on the Swedish west coast and only two specimens were
found in Pleuronectes microcephalus taken near Bergen, Norway. Both Odhner
and Nicoll reported the parasites as abundant in several species of fish, chiefly
flatfishes, and Odhner found it in P. microcephalus. Olsson reported the worms
from the stomach, whereas members of the family Zoogonidae are typically
parasites of the hindgut of fishes. Odhner regarded this observation of Olsson
as "ein ganz zufalliges Vorkommens oder als ein Irrtum." It is not impossible
that the specimens had developed in another host which had been eaten by
P. microcephalus and that they had migrated from the hindgut of their host
after it was eaten. Olsson's figure shows the opening of the acetabulum as oval,
longer in the transverse direction; in Odhner's figure the aperture is nearly
MORPHOLOGY AND LIFE HISTORY OF Z. LAEVIS
circular although the text states, "mit quergestellter, ovaler Lichtung." De-
scribing tlie acetabulum, Nicoll found the "aperture nearly circular, or if elliptical
the eccentricity is small." Olsson stated that the digestive ceca extend nearly
to the excretory vesicle, whereas Odhner and Nicoll reported that the ceca do not
extend past the middle of the acetabulum. In his figure, Olsson showed the
genital pore as lateral, near the level of the intestinal bifurcation, but unfortu-
nately there is no statement in the text and no legend to orient the figure; conse-
quently it is impossible to determine positively whether the opening is on the
right or left. Linton regarded Olsson's figure as a ventral view, with the genital
pore on the right side. This interpretation is probably correct since in other of
Olsson's figures, notably those of D. fasciatum and D. increscens in which the
genital pore is lateral, statements in the text show that the figures were drawn
from the ventral aspect. Furthermore, the protruding acetabulum of Z. viviparus
would make it exceedingly difficult for a fixed and stained specimen to lie on
the ventral side and consequently there are strong reasons for the opinion that
Olsson's specimen was mounted with the ventral side up. Both Odhner and
Nicoll, however, stated that in their specimens the genital pore is on the left side.
Nicoll's measurements of the miracidium are larger than those of Odhner, whose
figures agree with those of Olsson. In view of the disagreements in the descrip-
tions, there seems to be a reasonable doubt whether Odhner and Nicoll had the
same species that Olsson had described.
Linton accepted the accounts of Odhner and Nicoll as corrected descriptions
of Z. viviparus and distinguished Z. laevis from Z. viviparus on the "comparative
absence of spines" (a feature which he regarded as unimportant), the length of
the digestive ceca, and the location of the genital pore. Where Z. laevis differs
from the description of Z. viviparus as given by Odhner and Nicoll, it agrees
with Olsson's original description of the species. The American specimens,
described by Linton as Z. laevis, might reasonably be assigned to Z. viviparus,
but such disposition would imply that the specimens of Odhner and Nicoll
represent a different species, an inference that could not be justified without
restudying their material. A more complete description of Z. laevis and knowl-
edge of its life history will facilitate comparison with European species.
LITERATURE CITED
LINTON, EDWIN, 1940. Trematodes from fishes mainly from the Woods Hole region, Massa-
chusetts. Proc. U. S. Nat. Mus., 88: 1-172.
NICOLL, WILLIAM, 1907. A contribution towards a knowledge of the Entozoa of British marine
fishes. Part I. Ann. Mag. Nat. Hist., Ser. 7, 19: 66-94.
ODHNER, TH., 1902. Mitteilungen zur Kenntnis der Distomen. I. Ueber die Gattung Zoogonus
Lss. Zentr. Bakt., Parasit. u. Infekt., I, 31: 58-69.
OLSSON, P., 1868. Entozoa, iakttagna hos Skandinaviska hafsfiskar. Lunds Univ. Ars-skrifl,
4: pt. 2, (8).
STUNKARD, HORACE W., 1938. Distomum lasium Leidy, 1891 (Syn. Cercariaeum lintoni Miller
and Northup, 1926), the larval stage of Zoogonus rubellus (Olsson, 1868) (Syn. Z. mirus
Looss, 1901). Biol. Bull., 75: 308-334.
STUNKARD, HORACE W., 1941. Specificity and host-relations in the trematode genus Zoogonus.
Biol. Bull., 81: 205-214.
STUNKARD, HORACE W., 1942. The life cycle of Zoogonoides laevis Linton, 1940. Jour. Parasit.,
28 (Suppl.): 9-10.
INTERSEXUAL FEMALES AND INTERSEXUALITY
IN HABROBRACON
P. W. WHITING
(From the University of Pennsylvania, Philadelphia, and the
Marine Biological Laboratory, Woods Hole)
Much of the material discussed in this paper was collected by aid of a grant for assistance
from the Penrose Fund of the American Philosophical Society. The female intersexes were
found while work was being done under a grant from the Board of Graduate Education and
Research of the University of Pennsylvania.
Despite the great amount of breeding work especially centering about a
search for irregular sex types in the parasitic wasp Tlabrobracon juglandis (Ash-
mead), there has hitherto been reported (Whiting, Greb and Speicher, 1934)
only one real intersexual form. This is the mutant type gynoid, the gene for
which, gy, causes haploid males to be weakly intersexual. Gynoid females are
indistinguishable from wild type. The trait acts as a recessive in heterozygous
diploid males.
Gynoid males are similar to normal males in internal structure and in external
genitalia. Their ocelli are large resembling those of normal males. Their
normal male instincts indicate that the brain is structurally as in the male,
since mating reactions in Habrobracon are determined by the brain. Sclero-
tization of the abdomen is progressively heavier anteriorly, approximating the
condition found in the female. Antennae of normal males have about twenty
segments in the flagellum, those of females usually not more than thirteen. In
gynoid males the segments are reduced in number to that of the female, although
they are not quite as short and thick. Superficially a gynoid male suggests a
sex-mosaic or gyander with female head, male abdomen, but, as indicated,
certain structures are themselves intergrading, the body is approximately sym-
metrical with all parts presumably of the same genetic constitution and the type
is perpetuated as a pure-breeding form.
Nine intersexual females of the same species have recently been found and
are herewith reported for the first time. They occurred among the offspring of
a single female. Superficially these appear to be the reverse of the gynoid males,
being more masculine anteriorly, feminine posteriorly. The heads are character-
istically male having large ocelli and long antennae, flagellar segments ranging
from 18 to 21 with 20 as the mode. Tests made on five of the nine showed
indifference to caterpillars and vigorous attempts to mate with females, indicating
the brain to be structurally male. Abdominal sclerotization is male-like an-
teriorly. The first and second tergites are thin and the anterior sternal thick-
enings small. Sclerotization is progressively heavier posteriorly and sternal
thickenings become elongate, approximating the condition of the female.
Internal structures of the abdomen are as in the female, including normal
poison sack and glands and seminal receptacle. The ovaries, however, lack
differentiated nurse cells and ova. Each appears to be a pair of sacks of oogonia
238
INTERSEXES IN HABROBRACON
239
similar to the primordia of the ovarioles formed in the spun-in larva and normally
remaining essentially unchanged until the eyes of the pupa begin to turn black,
when differentiation of oocytes begins.
FIGURE 1. Gynoid male. Note the short "female" antennae and the heavy
anterior sclerotization of the abdomen. X 16.
FIGURE 2. I ntersexual female. Note the long "male" antennae and the decrease
in abdominal sclerotization anteriorly. X 16.
Like gynoid, these intersexes differ from sex mosaics in being approximately
symmetrical and similar to each other, in possessing sex intermediate characters
and in occurring in a group in one fraternity as if caused by an hereditary factor
rather than being scattered as single individuals.
240
P. W. WHITING
The fraternity containing these nine intersexes was small since the mother
had been discarded after eight egg-laying days, — two vials. Offspring were
being classified for sex and for certain eye colors. Nothing unusual was observed
in the first vial from which were obtained 13 females (+ 10, orange 3) and 11
males (+3, orange 8) of expected types. The nine intersexes (+7, orange 2)
were all found in the second vial which contained 13 males (+ 4, orange 9) and
no females. Proportion of wild type to orange eye color deviates from the
expected 1 : 1 in the females and intersexes in the opposite direction from that
FIGURE 3. Outlines of abdominal sternal thickenings and of external genitalia in normal male
(A), intersexual female (B) and normal female (C). X65.
in the males. This may be but a fluctuation due to small numbers or it may
indicate some chromosomal irregularity.
The males appeared structurally normal except that one had external genitalia
slightly reduced, a condition not infrequently found in Habrobracon. Dissection
of seven including this one showed internal genitalia normal. Flagellar segments
of antennae ranged from 18 to 21 with mode 20, normal for males. Ocelli were
of normal male size.
Since the offspring in the first vial had been discarded no tests could be
made. A mass culture from the vial 2 males, besides individual pairings of four
of these with related females, yielded nothing irregular in the immediate progeny
or in later generations. Many closely related side lines, which were being bred
for the eye color studies, gave only normal types. Unfortunately the eye colors
were brought into the mutant fraternity in such a way as to be of no significance
for determining which offspring were from fertilized, which from unfertilized
eggs.
IXTERSEXES IN HABROBRACON 241
»
DISCUSSION
The mother of these intersexes may have mated with two different brothers
before she \vas isolated for breeding. Sperm from one male may have been used
first to produce the females. The second male may have sired the intersexes
from sperm with a dominant intersex factor. This hypothesis is regarded as
unlikely in view of the fact that the females and intersexes were produced in
separate vials. In known instances of double matings the two types of offspring
expected eclose together, suggesting that the sperm have mixed.
If a mutation occurs in a primitive germ cell of a female of Habrobracon,
the resulting mutant tissue tends to form a stratum cutting across the two
ovarioles of both ovaries. This is due to the method of development from the
primitive germ cell mass which separates longitudinally into the gonad primordia
in a late embryonic stage. Each primordium elongates in the grown maggot,
the beginning of sex-differentiation, and is subsequently divided longitudinally
into two sacks, which become the ovarioles. Non-mutant tissue may then
function for a period producing eggs in the first vial, for example, while hetero-
zygous mutant tissue gives rise to eggs produced later.
If the intersexes be regarded as haploid (male) from unfertilized eggs, it
may be supposed that the mother's sperm supply was exhausted before transfer
to a second vial. Normal males and intersexual males would then segregate in
equal ratio in vial 2. This hypothesis is regarded as unlikely because of the
structure of the intersexes indicating that they are fundamentally female.
A dominant mutation in the ovaries should appear in only half of the zygotes
produced while the mutant tissue is functioning. Normal females might then be
expected in vial 2 at least equal in number to the intersexual. If, however, the
mutation occurred in the sex-differentiating chromosome segment as a modifica-
tion (deletion ?) in one of the sex factors (changing xb to xb1"), females (xa/xb)
might be replaced by intersexes (xa/xbm). Sperm, xa, fertilizing eggs from xa/xb
tissue in vial 1 would produce normal females, xa/xb, and diploid males, xa/xa,
but from xa/xbm tissue in vial 2, intersexual females, xa/xbm, and highly inviable
diploid males, xa/xa, would result. Unfertilized eggs would give haploid males
as expected but xbm males might be inviable.
The data must be regarded as inadequate to prove whether these intersexes
were due to a modification connected with the normally sex-differentiating factor
or whether, like gynoid, to an independent change. The series of sex alleles,
xa, xb, xc, etc., has been shown to be located at about the center of the left arm
of the linkage map. The gene gynoid, gy, is located near the distal end of the
right arm and therefore segregates independently of sex.
It is questionable whether the diverse effects of gynoid on antennae and
abdominal sclerotization should be regarded as multiple effects of a single gene.
Gynoid may possibly be a translocation from the differential segment determining
sex, the x factor. In a male with the sex allele in the normal position this might
give a complementary feminizing effect causing intersexuality.
Goldschmidt has defined an intersex as a phenotypic mosaic which begins
development as one sex according to its chromosomal constitution, XY or ZZ cT,
XX or WZ 9 , and then, after a turning-point, forms organs as in the opposite
sex. The earlier the turning-point, the higher the degree of intersexuality.
242 P. W. WHITING
With sex determination as in Habrobracon, haploid intersexes should begin
development as male, later shifting to female. The same should apply to
diploids if homozygous for the sex factor. Diploids heterozygous for sex should
begin development as female, later shifting to male.
The nine intersexual females discussed here must be regarded as more strongly
intersexual than gynoid males since antennae, ocelli and instincts are completely
sex reversed. The abdominal sclerotization of both intersexual types is inter-
mediate. Neither external nor internal genitalia are affected except that the
ovaries of the intersexual females fail to mature, remaining as sacks of oogonial
tissue. The turning-point appears to occur earlier in the anterior than in the
posterior region of the body or else the developmental processes forming the
external genitalia, which are begun before the turning-point, are such that they
must be carried to normal completion.
A comparison may here be made with triploid females of Habrobracon.
These are daughters of diploid males arising from fertilization of a normal egg by
a diploid sperm. They are presumably an unbalanced type, having two similar
paternal sex alleles combined with a dissimilar member of the series of maternal
origin. A diploid female may then be xa/xb, while a triploid will be xa/xa/xb
or xa/xb/xb. These formulae suggest the possibility of intersexuality, but
dominance relationships appear to be such that triploid females show no masculine
traits either in structure or in reactions.
Their egg production is, however, considerably lowered, being about one-fourth
that of diploid females. This reduction, involving both ova and nurse cells,
may be a small step toward intersexuality. A compensatory growth takes place
in the oogonial chamber which enlarges and elongates considerably under the
influence of feeding from host caterpillars. It would be of interest to know what
might have happened to the ovaries of the intersexual females if they had been
similarly nourished.
The suggestion has frequently been made that diploid males may be sex-
reversed females. This view originates in the older concept that diploidism as
such causes femaleness, a view now shown to be erroneous. Certain differences
of diploid from haploid males are to be expected dependent upon chromosome
number, but these are not necessarily in the direction of femaleness. Cell-size
for example is not only much greater in diploid males than in haploid : it actually
surpasses considerably the cell size of the normal diploid females. The sex-
linked gene "fused" causes antennal segments to be much reduced and fused
together. Fused females have much shorter antennae than haploid fused males.
Diploid fused males approximate fused females, having antennae only slightly
longer. Wild type females have much shorter antennae than haploid wyild type
males. Diploid wild type males approximate haploid, but, as determined by
count of segments, their antennae are slightly shorter. Difference in antennal
length of diploid from haploid males is due to chromosome number as such;
difference of females from diploid males is due to heterozygosis for the sex factor.
As regards antennae, the gene "fused" increases the difference due to chromosome
number, but tends to mask the difference due to sex. Intermediacy of diploid
males with respect to antennal length should not be regarded as intersexuality.
INTERSEXES IN HABROBRACON 243
SUMMARY
Nine female intersexes are described and compared with the one intersexual
form previously known in Habrobracon, the fertile mutant type gynoicl, a weakly
intersexual male.
These female intersexes proved sterile, having male heads and instincts and
abortive ovaries. They are, in general, female posteriorly, while gynoid males
have partially feminized heads but react like males.
Evidence suggests a dominant mutation in the sex-differentiating factor as a
possible cause of this female intersexuality.
On the basis of comparison with these female intersexes, it is suggested that
the structure of ovaries in triploid females represents a step toward intersexuality.
Antennal length of diploid males, both fused and wild type, although inter-
grading, is not regarded as due to intersexuality but to diploidy as such.
LITERATURE CITED
WHITING, P. W., RAYMOND J. GREB AND B. R. SPEICHER, 1934. A new type of sex-intergrade.
Biol. Bull., 66: 152-165.
NEW RECORDS OF SOME MARINE CHAETOPHORACEAE AND
CHAETOSPHAERIDIACEAE FOR NORTH AMERICA *
FRANCESCA THIVY
(University of Michigan, Ann Arbor, Michigan)
The study of the microscopic epiphytic, endophytic, shell-boring, and litho-
philic Chlorophyceae — a habit group not as yet fully explored for Woods Hole-
was suggested by Dr. Wm. Randolph Taylor and was carried out, under his
direction, during the summers of 1939 through 1942, at the Marine Biological
Laboratory. For helpful criticism the writer is greatly indebted to him.
In making this second report of the investigation, the writer wishes to express
her sincere gratitude to the Levi Barbour Foundation of the University of Michi-
gan, and Dr. Alma G. Stokey, through whose kindness these studies were made
possible.
Of the five members which are here added to the marine Chaetophoraceae of
North America, four have been described for Europe and one for the West Indies.
The present report of Diplochaete solitaria Collins (Chaetosphaeridiaceae) for
Woods Hole, Massachusetts, extends its distribution north of Jamaica.
CHAETOPHOREAE
Phaeophila Hauck, 1876. Plants endophytic, immersed within the external cell
walls or embedded in the cortex of the host, shell-boring, or rarely epiphytic;
thallus forming discs consisting of free or partly fused uniseriate branches;
branching lateral, alternate; cells cylindrical to round, often sinuous or with
irregular protrusions, frequently setigerous, with usually several nuclei; chloro-
plast parietal, plate-like, partly lining the cell wall, lobed at the margins, at
times becoming perforate or breaking up into discs; pyrenoids 1-13; setae usually
one to a cell, occasionally two or three arising from a cell, firm-walled, distinct,
very long, usually wavy but often straight while within the host and wavy
outside it, without a basal septum but often developing a thick collar at the base
and at times thereby becoming occluded, or, seta secondarily developing a basal
septum; sporangia intercalary, or terminal on branchlets, cylindrical, round or
conical, containing many zoospores, provided with a wide cylindrical neck;
sporangial neck twice the diameter of a seta but shorter, without wavy walls;
zoospores escaping together or one after the other, quadriflagellate; biflagellate
zooids observed in only one case (Huber 1892b, pp. 330-31); entrance into the
host effected by a germination tube arising from the anterior end of the zoospore;
zoospore and the lower end of the germination tube becoming empty and later
cut off by a septum.
* Paper from the Department of Botany, University of Michigan, No. 733.
244
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE 245
Phaeophila Engeri Reinke
Phaeophila divaricate, Huber, 1892b, p. 331.
Ochlochaete Engleri (Reinke) Hansgirg, 1892, p. 201.
Plant inhabiting living or dead shells of marine annelids and molluscs, im-
parting a grass green color to them, present on both sides or only on the dorsal side
of the shells often associated with other algae; thallus visible only after decalcifica-
tion, consisting of branched procumbent filaments extending in various directions;
cells occasionally showing one to several rhizoidal processes about 4.8 ^ diameter
(Plate II, figure 14); central cells deeply lobed, partly fused and forming a net-
work, but the filaments distinct peripherally; branching lateral; cells cylindrical
and either straight or sinuous along their length, or isodiametric with very
irregular lobes; cells 4.7-21.6/1 diameter, 1-5 times as long, when isodiametric
and irregular up to 52.4 n wide; in Spirorbis shells, however, cells only 4.7-11.8 ju
diameter, 1-5 times as long, when isodiametric and irregular up to 13.2 ju wide;
setae not numerous, sometimes lacking, either sinuous or straight, continuous
with the lumen of the supporting cell or secondarily separated by a cell wall
from it, both when basally open and when closed showing sparse granular con-
tents; setae when open at the base, often strengthened by a collar-like basal
thickening of the wall; width of setae 1.18-2.55/1. sometimes 3.6 M; length of
setae about 0.1 mm; cells 1-5 nucleate, with 1-7 pyrenoids; chloroplast parietal,
plate-like with lobed margins, at times breaking up into discoid portions, often
crowded with starch grains; cells frequently sending out 1-5 globular to oval,
vertical or lateral processes capable of developing an apical cap-like, stratified
swelling of the cell wall (Plate I, figures 1 and 2); or cells, in some instances,
bearing directly a similar or a peg-like thickening of the wall associated with a
seta or alone (Plate I, figures 9, 11, 15 and 16); end walls of cells occasionally
stratified; lateral walls sometimes having a number of small lentiform swellings
(Plate II, figures 7-10); sporangia flask-shaped to irregular, usually intercalary,
10.2-28.05 M long, 1-1^ times high, provided with a cylindrical neck about twice
the diameter of a seta, 3.53-5.32 n wide and the length 1-7 times the width;
zoospores 6-22 in number, ovoid, sometimes spindle-shaped, when swimming
measuring 3.6-8.4 y. in diameter, 1^-2 times as long, quadriflagellate, having a
pyrenoid, an eye-spot and a pair of contractile vacuoles; length of the flagella
usually equalling that of the zoospore; germination occurring with the formation
of a tube from the anterior end of the zoospore in a line with its long axis; the
zoospore and the lower part of the germination tube becoming empty and cut off
by a septum; aplanospores rarely seen, about 16 in a cell, each surrounded by a
cell wall, 7.11-9.41 ju diameter.
Woods Hole, Massachusetts: On clam shells (Mya arenaria L.) — Black Rock,
23 July 1941, coll. W. R. Taylor, July 1942; Great Harbor, 15 July 1941 and
14 Aug. 1942; Penzance, salt marsh, coll. Jennie L. S. Simpson, 26 Aug. 1942;
on quahaug shells (Venus mercenaria L.), Black Rock, 23 July 1941; on Busycon
carica Gmelin, Great Harbor, coll. W. J. Gilbert, 30 July 1941; on Polynices
duplicate, (Say), Spindle, 26 Aug. 1942; on Anomia simplex D'Orbigny, Spindle,
26 Aug. 1942; on Thais lapillus (L.), Gay Head, 16 July 1941; on Spirorbis
spirorbis (L.), attached to Fucus vesiculosis L., Spindle, 26 Sept. 1942; all ex-
cepting Spirorbis and Thais were dead shells.
246 FRANCESCA THIVY
Europe: On Spirorbis nautiloides — Kieler Fohrde, Engler and Reinke; Bulk,
Baltic Sea, Lakowitz; on Spirorbis and shells of various mussels and snails,
Kristineberg, Swedish west coast, Kylin; Weymouth, Dorset, Engl., Batters.
Distribution: Baltic Sea, southern coast of England, Atlantic coast of N. America.
Reinke, 1889, p. 86; Batters, 1902, p. 13; Migula, 1907, p. 807; Lakowitz, 1929,
p. 138, figure 194; Kylin, 1935, pp. 193-97, figures 3, A-F and 4, A-M.
P. Engleri is very similar to P. dendroides (Crouan) Batters, but is readily
recognized by its shell-inhabiting nature, the latter species being endophytic in
various marine algae. Reinke considered (1899, p. 86) that the sinuous cells of
P. Engleri distinguished it from P. dendroides. Though the latter also often
has deeply sinuated walls, a greater variability of cell shape and wall is character-
istic of P. Engleri. A cell of P. Engleri may bear several lateral and vertical
papilla-like processes which give it an extremely irregular contour (Plate I,
figures 1 and 2). As described by Kylin the processes may develop an apical
thickening of the wall, which breaks through the shell layers and communicates
with the external medium. When the papillate processes are lacking, evidence
of the tendency to form connections with the exterior is seen in the cap-like or
conical pegs formed directly on the cells as described above. P. dendroides was
found growing in the walls of Chondrus crispus (L.) Stackh., Polysiphonia flexi-
caulis (Harv.) Collins, and Champia parvula (C. Ag-.) Harv., at Woods Hole,
Mass. It does not have papillate cell processes, but its cells often have a solitary
PLATE I
Phaeophila Engleri Reinke
From Spirorbis
FIGURE 1. Filaments showing three papillate cell processes with apical thickening of the
wall, X 1000.
FIGURE 2. A cell with 5 processes resembling cells in Bor. et Flah., 1889, Plate VI, Figure 3;
other cells showing chloroplast, pyrenoids and starch grains, X 1000.
From Urosalpinx
FIGURE 3. Filaments in natural position, X 481.
FIGURE 4. Filament with a developing sporangium, X 481.
FIGURE 5. Cell filled with starch grains, X 681.
FIGURE 6. A terminal sporangium, X 481.
From Mya
FIGURE 7, a-e. Zoospores; a, living, showing chloroplast and eye-spot; b-e, stained with
iodine; d, unusually large zoospore with 2 contractile vacuoles, eye-spot and pyrenoids, X 929.
FIGURE 8. Intercalary sporangium with aplanospores, X 710.
FIGURE 9. Filament showing a sporangium with a peg on the wall, by the side of its neck.
X 481.
FIGURE 10. Filament showing a sporangium with a seta beside the neck, X 471.
FIGURE 11. Sporangium with a peg and a narrow neck, X 1000.
FIGURE 12. A globular sporangium, X 481.
FIGURE 13. Empty sporangia, X 763.
FIGURE 14. Two irregular cells embedded deeply in the shell, X 734.
FIGURE 15. Cell with a seta and a dome-shaped tubercle on the wall, X 1000.
From Polynices
FIGURE 16. Cell with a seta and a tubercle on the wall, X 1000.
FIGURE 17. Sporangium with zoospores showing eye-spots and pyrenoids, X 547.
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE
247
248 FRANCESCA THIVY
lateral process resembling the initial stage of a branch; the peculiar swellings of
the wall found in P. Engleri are completely absent.
Another difference between the two species is discernible in their sporangia.
In both they are usually intercalary, cylindrical to flask-shaped, but the length
of the emission tube is much shorter in P. Engleri (Kylin, figure 4L) than in
P. dendroides (Huber, Plate XVI, figure 9). It is either a short papilla or a tube
in length about 1-2 times the height of the cell in the first case, and about 4
times the height of the cell in the other. In P. dendroides it extends beyond the
host to about 25.5 ju as seen in the examples at Woods Hole, but in P. Engleri
the tube apparently does not project beyond the shell surface. It is significant
that figure 4L cited above represents sporangia in a culture, without the shell,
as it affords evidence that the short emission tube is a stable character of the
species.
The phenomenon of setae secondarily developing a basal wall found in
P. Engleri may also be seen, though very rarely, in P. dendroides.
That chalk-boring algae are of greater importance than animals of similar
habitat in breaking down calcareous substrata and releasing potassium, mag-
nesium and other elements, is stated by Nadson (1927, p. 153). He says that
various blue green algae as well as Gomontia polyrhiza (Lagerh.) Born, et Flah.
and Ostreobium Queketti are widely distributed, but not P. Engleri and Conchocelis
rosea Batters. One may conclude from the common occurrence of P. Engleri
at Woods Hole and presumably also in Europe, the alga probably is present in
many more localities than are so far known. In all cases of clam and quahaug
shells examined for P. Engleri, at Woods Hole, the latter alga was mixed with the
large unicells of Gomontia, but in Anomia, Polynices, Thais and Spirorbis,
P. Engleri was present alone.
PLATE II
Phaeophila Engleri Reinke
From Mya
FIGURES 1-2. Cells showing setae with the secondary basal septum; in Figure 2 cell contents
seen in the seta; Figure 1 X 1000; Figure 2 X 592.
FIGURE 3. Cell showing seta with a collar-like basal thickening of the wall and cytoplasmic
granules, X 1000.
FIGURES 4-5. Cells showing basally open setae, Figure 4 X 493; Figure 5 X 592.
FIGURE 6. Cell with two setae, one basally open, the other with a basal septum, X 592.
FIGURES 7-10. Cells showing lenticular swellings in their lateral walls, Figure 7 X 751;
Figure 8 X 586; Figure 9 X 624; Figure 10 X 724, from a culture on shells.
FIGURE 11. Filaments showing narrow cylindrical, and large globular cells, X 666.
FIGURE 12. Filament showing irregular cells resembling in shape and size some of the cells
in Bornet and Flahault, 1899, Plate VII, Figure 16; X 1142, from a culture on shells.
FIGURE 13. From a culture on shells, an intercalary cell showing a large cylindrical process
resembling in size the spindle-shaped cell process in Taylor, 1937, Plate I, Figure 13, and agreeing
in size as well as shape with cell processes in Bornet and Flahault, /. c., Plate VII, Figures 14
and 16; X 703.
FIGURE 14. Cells with rhizoids as in /. c., Plate VI, Figure 7; X 634.
From Anomia
FIGURE 15. Filament showing several nuclei to a cell, as in /. c., Plate VIII, Figure 20;
X 813, from a culture on shells.
From Urosalpinx
FIGURE 16. Filaments with two flask-shaped young sporangia, X 813.
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE
249
PLATE II
250 FRANCESCA THIVY
Because of the frequent occurrence together of Gomontia polyrhiza and
Phaeophila Engleri, the swollen cells of the latter could be taken to represent
the intermediate stage in the development of intercalary cells into the unicells of
Gomontia, for which such an origin has been described by Bornet and Flahault
(1888, pp. 163-65; 1889, pp. CLII-CLX, Plates VI-VIII). Observations of
P. Engleri and G. polyrhiza made in the course of this study and a comparison
of these algae (cf. Plate II, figures 12-15) with the descriptions and figures of
G. polyrhiza given by Bornet and Flahault (op. cit.) for the Atlantic coast of
France and the Mediterranean Sea and by Taylor (1937, pp. 57-58, Plate I,
figure 13) for the Atlantic coast of North America, suggest that the filaments
attributed to Gomontia are, with little doubt, the filaments of Phaeophila Engleri.
The latter alga has probably often been overlooked and its distribution must be
at least as wide as that of Gomontia polyrhiza.
P. divaricata Huber (p. 331, Plate XVI, figures 12 and 13) agrees with P.
Engleri in cell size and in its calciphilous habit within the encrusted walls of the
'stems' of Acetabularia. The sporangia of the two species are very similar,
and characteristically different from those of P. dendroides; the form on Acetabu-
laria is probably the same as P. Engleri Reinke. It is of interest also to note,
since P. Engleri has evidently been taken to be a stage of Gomontia, that Huber
says some of the cells of P. divaricata are swollen toward their summits and
resemble the habit of Gomontia.
P. dendroides var. calcicola Hansgirg growing in the shells of gastropods and
also in Corallina and Lithothamnium is reported for Istria and Dalmatia
(Hansgirg, 1892, p. 201). Taking into account Kylin's remark (p. 194) that
P. Engleri is .more common at Kristineberg on the shells of various mussels and
snails than on Spirorbis, it appears likely that P. dendroides var. calcicola is
synonymous with P. Engleri.
The present observations show that the forms of Phaeophila, occurring on
Spirorbis on the one hand, and in the shells of molluscs on the other, cannot be
considered as different species as they vary only in cell size. The smaller di-
mensions of the perforating form on Spirorbis may result from space limitation.
Ectochaete (Huber) Wille, 1909. Species marine or fresh-water; plants
microscopic, endophytic in other algae; thallus usually forming discs of radiating
partly fused filaments within the host wall, in one species developing pseudo-
parenchymatous cushions after emerging from the host wall, in another anchored
in the mucilage investing the assimilatory filaments of the host in the form of
threads interwoven with those of the host; branching lateral or rarely dichoto-
mous; cells uninucleate, cylindrical to round, setigerous; seta solitary, straight,
very long, showing sparse granular contents, without a basal septum but at
times the lumen not visible at its base; chloroplast single, parietal, incompletely
cylindrical, plate-like or sometimes perforate, usually with numerous starch
grains; pyrenoids 1-8; plants reproducing by zoospores or isogamous gametes;
zoosporangia or gametangia developing from vegetative cells, having a short to
long beak and containing many zooids; zoospores and gametes biflagellate, with
a chloroplast, a pyrenoid and usually an eye-spot; germination either accom-
panied by the production of a germination tube from the anterior end of the
zoospore or not.
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE 251
Ectochaete leptochaete (Huber) Wille. Plant filamentous, endophytic in
the external cell walls of green, brown and red algae, forming minute mono-
stromatic discs 64-166 /z diameter; branches lateral, irregularly alternate, arising
as a rule near the proximal end of a cell, often with the basal septum occurring
a short distance above the subtending cell, in the older stages showing fusions
in the center of the disc but filaments remaining free outwardly; central cells
with a diameter of 5. 88-11. 82 /z (according to Huber, 5-15 ju), isodiametric or
1.5 times as long; cells towards the ends of the filaments usually 3.5-4.7 n di-
ameter, 2-4 times as long; setae very delicate, tubular, containing cytoplasmic
granules, 1.2-1.5 ju diameter, about 80 ju in length, with a constriction at the
point of exit from the host wall; lumen of seta continuous with that of the sup-
porting cell but sometimes not visible at its base, the wall being opaque at this
point; chloroplast plate-like, incompletely lining the wall, perforate in the older
stages; pyrenoids usually 1-3, sometimes 4; many central cells serving as zoospo-
rangia, 5.88-11.82 /i diameter, round, conical or shortly cylindrical with a short,
vertical, hyaline beak, in length usually one third the diameter of the cell and
rarely half the diameter; zoospores about 12 in each, 2.4/1 diameter, either
spherical or ovoid, 4-5 n long, biflagellate, with flagella three times the cell
length; during germination the anterior end of the zoospore prolonging into a
germination tube and entering the host, the empty zoospore not cut off by a
wall (Huber 1892b, Plate XV, figures 8 and 9).
Woods Hole, Massachusetts: In the external walls of Polysiphonia novae-angliae
Taylor, especially in the basal part of the host, in the wash at Nobska Point,
September 1 and September 10, 1942; in Chondrus crispus (L.) Stackh. (inter-
tidal) and the leaves of Sargassum Filipendula C. Ag., from the wash at Nobska
Point, 10 September 1942.
Huber recorded the species for the Gulf of Lyons where it was present in
salt water ponds, on Cladophora, Chaetomorpha Liniim and Ceramium diaphanum
(Lightf.) Roth, (Nov. and April), and for the Bay of Biscay, on a Chaetomorpha
(Sept.). It is cited by Feldmann for the Gulf of Lyons, as growing in Dictyota
dichotoma and Dilophus Fasciola, (May to June). For the English Channel, we
have reports by Hariot, on Cladophora tenerrima at Tatihou, by Batters from
Devon, and by Newton, on Ectocarpus penicilliformis, Ceramium diaphanum
and Cladophora, also from Devon.
Distribution: W. Mediterranean, Atlantic Coasts of France, England and N.
America.
Huber, 1892b, pp. 319-26, Plate XV, figures 1-9; Batters, 1902, p. 14; Wille,
1909, p. 79; Oltmanns, 1922, I, pp. 299-300; Printz, 1927, p. 194; Hamel, 1930,
p. 28; Newton, 1931, p. 62; Feldmann, 1937, p. 181; Thivy, 1942, pp. 98 et seq.
The species may be distinguished from E. Taylori, another marine species
to which it comes nearest in structure, by its habit and by the delicate setae
only half as wide as those of E. Taylori, which has setae 2.66-3.8 n in diameter,
as well as by its smaller cell size, for in E. Taylori the cells have a diameter of
8-18 n and sometimes up to 25.5 p. Besides, each of the two species is char-
252 FRANCESCA THIVY
acterized by its sporangium and by its method of germination. The careful
and detailed description given by Huber 1892b applies closely to the alga as found
at Woods Hole, except that the cell diameter does not reach 1 5 // in the present case.
In cultures without the host, grown in sterile 0.875 Detmer's solution, the
species behaves, on the whole, like E. Taylori, forming a disc from which upright,
branched filaments arise in the place of the setae and grow into a fuzzy mass
about 1 mm. in diameter. The vegetative cells and sporangia are larger than
in nature and reach 23.52 /j. diameter. The zoospores are also larger, 3.5-5.1 M
in diameter, and there are up to 30 in a sporangium. When the culture is trans-
ferred to plain sterile sea water, setae appear after the lapse of a week. The
basal constriction observed by Huber both in nature and in cultures of the
endophyte still within the host, is seen in the above cultures, though the host
is absent.
Ectochaete vagans (Borgesen) comb. nov. (Endoderma vagans Borgesen).
Thallus filamentous, microscopic, endophytic in the external cell walls of various
algae, creeping; filaments uniseriate, forming a network or radiating from a
center, fused to a slight extent in the middle of the thallus or free throughout,
widely spreading at their ends; branches lateral, irregularly alternate, sometimes
without a basal septum; cells sub-cylindrical, often having at the middle or at
the upper end a lateral obtuse outgrowth remaining as such or growing into a
branch, 5.32-13.3 n diameter, 1-4 times as long, with the diameter at an out-
growth usually doubled; end cells of attenuate filaments 1.23-5.32 /x and 5-17
times as long; occasionally intermediate cells may be narrower and longer than
normal; plants having on their walls, either laterally or vertically, dome-shaped,
conical or peg-like tubercles in width a third to half the diameter of the cells,
but lacking them sometimes; an extremely fine, long, straight seta, 1.176/1 in
diameter, about 100 /x long, proceeding from the conical tubercle; seta continuous
with the lumen of the supporting cell, tubular but the lumen usually not visible
and when in view seen only above the opaque base of the seta, deciduous; conical
base of seta about 8 M diameter, embedded in the host wall, incrassate, usually
transversely striate or punctate with less refractive areas; chloroplast parietal,
PLATE III
Ectochaete leptochaete (Huber) Wille
From Polysiphonia novae-angliae Taylor
FIGURES 1-2. Habit of two plants, Figure 1 X 749; Figure 2 X 855.
FIGURE 3. Sporangia (necks not visible) and uninucleale cells in nature, in surface view,
X 855.
FIGURE 4. Habit of plant in culture (0.875 Detmer solution) showing branches in the place
of setae, X 24.
FIGURE 5. Filament from a culture (0.875 Detmer solution) bearing a long two-celled erect
branch in the place of a seta, X 394.
FIGURE 6. Terminal colorless cell ending in a basally open seta (sea water culture), X 855.
FIGURE 7. Terminal green cell ending in a basally open seta (sea water culture), X 855.
FIGURE 8. Intercalary cells with setae showing continuity of the lumen from cell into seta
(sea water culture), X 855.
FIGURE 9. Empty sporangia with rather long necks (culture in 0.875 Detmer solution
+ algal extract), X 394.
FIGURES 10-11. Sporangia with necks of average length filled with a semi-hyaline wall
substance. (Culture in 0.875 Detmer solution + algal extract), Figure 10 X 444, Figure 1 1 X 394.
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE
253
PLATE III
254 FRANCESCA THIVY
partly lining the cell wall, in older cells obscured by the numerous starch grains,
appearing bright green in dry specimens; pyrenoids 1-5, usually 1-3 (Borgesen
found 5-7); sporangia arising from usually somewhat enlarged vegetative cells,
producing 7-15 zoospores; the latter 2-3 n diameter, 4-4.7 p. long; in the sporangia
provided with tubercles the opening occurring through them; the number of
flagella not observed so far; sexual reproduction not known; during germination
the anterior end of the zoospore growing in a line with its long axis and forming
a germination tube, the latter becoming the first green cell.
Woods Hole, Massachusetts: In the wall of Polysiphonia flexicaulis (Harvey)
Collins, coll. Gladys Bulmer, in the wash at Little Harbor, Aug. 31, 1940; also
of P. Harveyi Bail. v. Olneyi (Harv.) Collins, coll. Jennie L. S. Simpson, in the
wash at Nobska Point, Sept. 9, 1942.
The alga was found by Borgesen growing at a depth of 5 fathoms on Griffithsia
globulifera Harv., near Buck Island, St. Croix, Virgin Islands. It was again
collected by Taylor, growing in the cell wall of old specimens of Bryopsis washed
ashore in Rockly Bay, Tobago Island, Br. West Indies. Thus the alga occurs
in the sublittoral region and probably also at intertidal levels.
Distribution: West Indies, Atlantic Coast of North America.
Borgesen, 1920, pp. 418-19, figure 400; Taylor, 1942, pp. 15-16.
In the specimens collected at Woods Hole in 1942, while observing the cellulose
tubercles, the presence of very delicate setae was noticed for the first time in
this species. Borgesen's figures, especially 400c and d, agree with the appearance
of the tubercles seen in the above specimens, but he was of the opinion that
the structures in question on his plants were small cells. He remarks that now
and then five to six or more narrow bodies, lying above each other, are present
in them. He also says these bodies are filled with starch and leaves it to further
observations on living specimens, as he had studied only dry ones, to add infor-
mation about them. Though Borgesen does not refer to the cellulose tubercles,
PLATE IV
Ectochaete vagans (Borgesen) comb. nov.
On Polysiphonia Harveyi Bail. v. Olneyi (Harv.) Collins
FIGURE 1. Habit of endophyte within the host wall; no tubercles or setae seen, X 75.
FIGURE 2. Thallus showing fusions in the center and lacking tubercles and setae; two open
sporangia seen, X 352.
FIGURE 3. Two young plants; a tubercle seen at a, X 352.
FIGURE 4. Lateral view of two filaments within the host wall; setae with conical incrassate
bases, X 749.
FIGURE 5. Germling showing the germination tube dividing into two cells and the empty
zoospore, X 1649.
FIGURE 6. Two-celled germling embedded in the host wall with the empty zoospore on
the surface of the host, X 666.
FIGURE 7. Three-celled germling, X 465.
On Polysiphonia flexicaulis (Harv.) Collins
FIGURE 8. Plant showing reticulate habit, tubercles, sporangia and empty sporangia,
X 273.
FIGURE 9. Filament showing three sporangia. Two with the opening in the center of the
tubercle, X 431.
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE
255
PLATE IV
256 FRANCESCA THIVY
the figures cited above probably depict these structures, considered by him as
elevations of cells cut off by a cell wall.
In the living examples the elevations or outgrowths of cells are distinct from
the cellulose tubercles. The former show the presence of large amounts of
starch, becoming black like the rest of the cell contents when treated with
Schultz's solution. Occasionally the apex of an outgrowth develops into a
cellulose tubercle (Plate V, figures 12 and 13), which takes on a violet color with
the above reagent. The tubercles that also occur directly on the cells do not
contain cell contents, but consist entirely of wall substance.
The presence of setae in the collection from Woods Hole leaves little doubt
that the conical tubercles are the persistent bases of the setae, while the dome-
shaped and peg-like ones appear to be structures that are either sui generis, or
setae arrested in their development. In common with the setae of other endo-
phytic Chaetophoraceae, those of the present alga presumably function in
creating a large surface of contact with the medium external to the host; the
persistent bases of the setae as well as the other tubercles very likely subserve
the same role.
The setigerous character of the species necessitates its transfer from Endo-
derma Lagerh. to Ectochaete (Huber) Wille.
The habit of Phaeophila and Ectochaete may be looked upon as reduced
(specialized) and being derived from a heterotrichous habit in which the erect
system has been replaced by setae, or it may be considered relatively primitive,
and as evolving from a simpler entirely procumbent habit (Fritsch, 1942, p. 401).
Erect filaments developing instead of setae in cultures of these two genera may
have not only physiological but also phylogenetic meaning (Huber, 1892a, p. 333).
The setae of Ectochaete leptochaete, E. vagans and Phaeophila Engleri
A seta continuous with the lumen of the supporting cell is said to be char-
acteristic of the genus Ectochaete (Huber, 1892a, p. 331, figure 5; Printz, 1927,
p. 194). In the present cultures of E. leptochaete the setae show open bases in
favorable examples; in others the bases look solid, the setae appearing to take
PLATE V
Ectochaete vagans (Borgesen) comb. nov.
On Polysiphonia Harveyi Bail. v. Olneyi (Harv.) Collins
FIGURES 1-3. Filaments with tubercles on the wall; in Figure 1 the broken line representing
the host wall X 1000.
FIGURES 4-5. Cells showing the bases of the, setae, X 1000.
FIGURE 6. At a, seta with an opaque base, showing the lumen in the upper part; at b,
a peg-like tubercle on the wall, X 1000.
FIGURES 7-8. Long terminal cells of two filaments, X 1000.
FIGURE 9. Filament with two sporangia, one showing a tubercle, X 1000.
FIGURES 10-11. Germlings showing the empty zoospore outside the host wall and the
germination tube within; Figure 10 X 764; Figure 11 X 1000.
FIGURE 12. Filament showing a cell bearing a process with an apical thickening of the
wall at a, and a peg-like tubercle on the wall at b, X 1000.
On Polysiphonia flexicaulis (Harv.) Collins
FIGURE 13. Filament showing a cell process with an apical thickening of the wall at a,
and an open sporangium at b, X 575.
FIGURE 14. Three Sporangia, each with a tubercle, X 575.
( HAETOPHORACEAE AND CHAETOSPHAERIDIACEAE
257
PLATE V
258 FRANCESCA THIVY
their origin from the cell wall, however the lumen is clear along the rest of the
length of the seta and it contains granules especially at the apical part. A
rather similar condition is present in the genus Chaetosphaeridium.
Klebahn (1892, p. 269, Plate IV) considers the setae of C. minus Hansg.
(C. Pringsheimii Klebh.; Aphanochaete globosa [Nordst.] Wolle var. minor Hansg.,
cf. West, 1904, p. 182-83) as agreeing in structure with those of Coleochaete,
and his figures show continuity of the lumen from cell to seta, while Mobius
(1892, p. 104, figure 8) says with reference to the setae of the above alga (for
synonymy cf. Hazen 1902, p. 228) ". . . sondern hier findet sich eben nur die
Kommunikation durch Verdickung der Membran sehr verengt." Huber (/. c.,
pp. 338-39) pointed out that the setae of Chaetosphaeridium globosum (Nordst.)
Klebh. (Aphanochaete var. a globosa Hansg.) are transitional between tubular
and solid setae, since their lumina are extremely narrow with protoplasm repre-
sented by only a few granules, and Oltmanns (1922, I, p. 303) cites the same for
the species. On the other hand, Wille (1909, p. 100), Heering (1914, p. 144),
Printz (1927, p. 233) attribute solid, homogeneous, tegumentary setae to the
genus, and Fritsch (1935, p. 286) considers that setae of this type characterize
the family. The lumen is so greatly reduced in the genus that both these
interpretations exist.
The difficulty of seeing the passage between the seta and its supporting cell,
because of the thickness of the wall and the narrowness of the lumen at the base
of the seta, may give rise to the view that the setae are tegumentary in E. lepto-
chaele, but it is possible in some cases to see clearly the passage from the cell
into the seta, and this confirms Huber's description of them.
The setae of Ectochaete vagans and Phaeophila Engleri are comparable with
those of Acrochaete repens Prings. At Woods Hole, in certain cells of Acrochaete
the lumen of the seta could be seen connected with that of the cell (Plate VI,
figure 1, a and c) while in most cases the seta is refractive and cannot be viewed
in optical section (Plate VI, figure 1, b and figure 4); in a few instances it was
found also that a septum may arise at the base of a seta, separating it from the
cell, and in these examples the setae had been shed, breaking a little above
their bases (Plate VI, figure 3). Huber (I.e., p. 328, figure 3, b) considers such
septa as secondary developments since the setae lack nuclei of their own.
The opaque conical base of the seta in Ectochaete vagans similarily cannot be
seen in optical section, but since a lumen is visible at times above the base of
the seta (Plate V, figure 6a) a connection probably exists between seta and cell
as in Acrochaete.
The basally open and the basally septate setae of Phaeophila Engleri agree
with the corresponding setae of Acrochaete, and as in the latter the formation
of a basal septum in certain of its setae is presumably secondary.
It is of interest to note that the cells of Acrochaete may have cellulose tubercles
as in both the species compared with it above, but in it invariably found at the
apices of cell processes (Plate VI, figure 2).
LEPTOSIREAE
Entocladia Reinke, 1879. Algae forming discs in the cell wall, immersed in the
mucilage of the host, penetrating the tissues of the host, or found in the cuticular
layer of Coelenterata and Bryozoa and in the shells of molluscs; thallus of radi-
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE 259
ating filaments often subparenchymatously congested in the center; hairs and
setae absent; cells cylindrical to isodiametric, sometimes irregularly swollen,
uninucleate, with a parietal chloroplast and one to several pyrenoids; sporangia
appearing in large numbers, sometimes slightly larger than vegetative cells,
provided with a papilla-like or short, tubular beak, producing 8-28 usually
quadriflagellate zoospores and exceptionally biflagellate zoospores and isogametes;
germination of zoospore endophytic or epiphytic in type.
Entocladia testarum Kylin. Alga inhabiting the superficial layers of the dead
shells of molluscs, in shallow water and salt marshes; thallus forming a more or
less' spherical mass of interlacing filaments; internal filaments partly fused,
consisting of round to oval or irregular shaped cells 7.1-10.58 /j. diam. and of
cylindrical cells 3.52 /JL diam., 3-8 times as long; in the intermediate region of
the thallus one-celled, oval, decumbent branchlets present, with a diameter of
9.4 n; outer filaments of the semispherical mass free, with cylindrical cells about
3.53 /i diam., 3-8 times as long, bearing one to several decumbent or vertical,
papilla-like processes 3.53-5.88 ju diam.; tips of outer filaments bending upwards
towards the surface of the shell; cells with a parietal, plate-like, sometimes perforate
chloroplast; starch grains often numerous, making the chloroplast appear reticu-
late; pyrenoids 1-5, usually 2 or 3; sporangial cells cylindrical to club-shaped or
round 7.06-12.94 ju diam., and neck 4.71-5.88 \i diam., 7.1-9.41 n long; zoospores
4-14 in each, 2.35-4.1 // diam., twice as long, pear-shaped, quadriflagellate, with
flagella equalling the cells in length, with an eye-spot, a chloroplast, a pyrenoid,
and starch grains; germination of the epiphytic type, not involving the formation
of a germination tube.
Woods Hole, Massachusetts: Rich growth in the empty shells of Mya arenaria
L. along with traces of Gomontia polyrhiza and Phaeophila Engleri, Penzance
salt marsh coll. Jennie L. S. Simpson, August 29, 1942.
Europe: Kristineberg, Swedish west coast, very abundant on dead Mya arenaria
shells, Kylin.
Distribution: Baltic Sea; Atlantic coast of North America.
Kylin, 1935. pp. 197-201, figures 5 A-R; 6 A-F.
Kylin gives an account of the characteristics of the alga in cultures, both
isolated and on shells. In the former case spherical masses of filaments were
formed with cells larger than in nature, and with the length of sporangial necks
reaching twice the diameter of the sporangia (Kylin 1935, figure 5 P and R)
that is, a little longer than when within the shell. When a culture was grown
on Mya arenaria shells, some of the filaments emerged from it, and these were
10-15 n wide while the immersed filaments were 5-10 /x wide; sporangia were
obtained under both conditions.
Kylin points out (L c., p. 203) that the species is difficult to distinguish from
E. tenuis Kylin and that the two usually are found together, with Gomontia
and Phaeophila; but he mentions that the cells are narrower and longer in E.
tennis, (the measurements being 3-5 fj. diam., 8-20 times long in the young cells
260 FRANCESCA THIVY
and 5-8 p diam., 4-8 times long in older ones). Since no cells in E. testarum as
met with at Woods Hole, in agreement with Kylin's description, exceed the
length of 8 times their diameter, and because of the characteristic cushion habit
with numerous decumbent or erect branchlets, cell processes, and branch tips,
found in nature in the present specimens, but seen by Kylin only in his cultures,
the species appears to be singular and unmistakable. Moreover a definite
difference between the latter two species is to be seen in their germlings (7. c.,
p. 204) as E. testarum, unlike E. tennis, lacks a germination tube.
Regarding the phylogenetic significance of the plagiotropic dendronema or
nematoparenchyma (terms ex Schussnig, 1938) under which types the Leptosireae
come, Fritsch postulates: ". . . the first step in the evolution of the heterotrichous
habit may have been a branched creeping filament or expanse attached throughout
to the substratum, ... a possibility not incompatible with the existence of
prostrate types that have arisen by reduction from a heterotrichous filament."
ULVELLEAE
Ochlochaete Thwaites, 1849. Plants epiphytic on marine phanerogams and
algae, or creeping on stones, shells and debris, green or olive-green in color;
thallus filamentous, but more or less pseudoparenchymatous from the beginning;
branches subdichotomous, either (O.fero.v, 0. lentiformis, 0. gratulans) superposed
PLATE VI
Acrochaete repens Prings.
FIGURE 1. Filament showing at a and c continuity of the lumen between cell and seta
at b the opaque base of a seta, X 528.
FIGURE 2. Filament with vertical cell processes, each with a tubercle at the apex, X 329.
FIGURE 3. A cell showing a secondary septum between seta and cell process, X 947.
FIGURE 4. Cell with seta showing lumen above and opaque base below, X 528.
Entocladia testarum Kylin
From Mya
FIGURE 5. Superficial cells of the thallus showing horizontal filaments, erect processes and
unicellular branches, X 991.
FIGURE 6. Terminal cell with two erect processes, X 991.
FIGURE 7. Filament with a decumbent apex, a lateral process, a decumbent lateral process
at a, and a vertical process at b, X 1142.
FIGURE 8. A horizontal filament from the intermediate region of the thallus showing a
unicellular decumbent branchlet, X 1142.
FIGURE 9. Filament showing two uninucleate cells and the decumbent tip of a process,
X 1142.
FIGURE 10. Unicellular branch showing nucleus, chloroplast and pyrenoid, X 1273.
FIGURE 11-13. Intercalary sporangia showing necks, X 1142.
Ochlochaete lentiformis Huber
FIGURES 14-15. Young discs from Busycon, X 529.
FIGURE 16. Cells from a disc growing on a stone showing arrangement in three fused layers,
X 529.
FIGURE 17. Cells from a disc growing on a stone showing two fused layers, X 529.
FIGURE 18. Marginal cell of a young disc showing the basally open seta, from Busycon,
X 259.
FIGURE 19. Seta with the lumen narrowed at the base, from a stone, X 1142.
FIGURE 20. Two-celled germling from Busycon, X 2283.
FIGURE 21. Transverse section of thallus showing sporangia, one of them with zoospores,
X 800, figure from Huber, 1892b.
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE
261
PLATE VI
262 FRANCESCA THIVY
and completely fused, resulting in a 2-3-stromatic disc, or (O. hystrix) in one
plane and united only in the centre of the thallus; some of the cells bearing a
seta; seta thick walled, firm, straight, showing sparse contents, with lumen
continuous with that of the supporting cell; cells round to rectangular, uni-
nucleate; chloroplast parietal, plate-like, with usually one and sometimes 2-3
pyrenoids, often with numerous starch grains; reproduction by 4-flagellate
zoospores, produced in large numbers in sporangia provided with a short neck;
zoospores ovoid with a hyaline beak and a pyrenoid, escaping in a mass and
expelled with force; zoospore germinating by growing at right angles to its long
axis, i. e. epiphytic in type.
Ochlochaete lentiformis Huber. Alga forming green specks on the surface of
stones, shells and debris; disc 51-831 ^ in diam., more or less circular, pulvinate,
consisting of filaments fused into a firm tissue, without intercellular spaces,
2-3-stromatic in the center, monostromatic at the periphery; short erect branches
of one or two cells in length arising near the margin of the disc, making it poly-
stromatic; margin of disc truncate, or showing free ends of filaments to a depth
of 1-2 cells; cells often exhibiting a radial arrangement in the disc, 5.1-12.75 ju
diam., sometimes up to 22.95 n diam., some of the superficial cells of the disc
at times only 3.5 IJL diam.; cells at the margin in old discs round or oval with the
radial axis longer, in young discs as above and also sometimes when oval with
the long axis in the tangential direction; superficial and marginal cells often
bearing a seta; setae 2.35-3.82 p. diam. near the base, 0.85-1. 8 ;u near the tip,
about 0.5-1 mm. long; lumen of seta and supporting cell confluent; chloroplasts
plate-like, with 1-3 pyrenoids; sporangia with a short neck, forming about 16
zoospores expelled together explosively, germinating without producing a germi-
nation tube.
Woods Hole, Massachusetts: On old dead shell of Busycon carica Gmelin,
Great Harbor, coll. W. J. Gilbert, July 30, 1941; on dead shells of Polynices
duplicata (Say), on dead shells of Anomia simplex D'Orbigny, on broken bits
of porcelain, on white pebbles, all at the Spindle, August 26, 1942.
Europe: Croisic, Bay of Biscay, abundant on pieces of porcelain, old pipes and
glass, Huber, September 1891.
Distribution : Atlantic coasts of France and North America.
Huber, 1892b, pp. 296-97, Plate XI, figures 1-3; Wille, 1909, p. 88; Printz, 1927,
p. 211; Hamel, 1930, pp. 44 and 46.
This alga may pass for Protoderma marinum Reinke, associated with which
it was seen on stones, pieces of porcelain and shells in the present collections.
They are alike in forming pseudoparenchymatous discs several layers in thickness,
but they are distinguished by the presence or absence of setae. When setae are
few in 0. lentiformis it can be distinguished easily by the size of its cells, for in
P. marinum the cell diameter is about a half of that of the former and is 3.5-
7.65 n, some of the central cells occasionally reaching up to 12 ju. The central
cells in both are more or less round though they may be isodiametric-angular in
CHAETOPHORACEAE AND CHAETOSPHAERIDIACEAE 263
Protoderma marinum. The marginal cells are round or oval in both, but some-
times oblong in P. marinum.
O. ferox Huber (/. c., pp. 292-93, Plate X, figures 1-10) is known for Massa-
chusetts (Collins, 1909, p. 288; Taylor, 1937, p. 55). It differs from the present
species in choice of substratum, being epiphytic on Cladophora, Chaetomorpha
and Zostera, as well as by the larger size of its thallus and cells, and the presence
of setae in greater numbers.
Fritsch (1935, p. 260) considers the upper layers as possibly formed by short
erect adpressed branches, while Huber described them as arising from superposed,
horizontally growing branches. Both interpretations are tenable, since the
branches are not long enough to see whether they remain erect or grow parallel
to the surface of the disc. The habit of Ochlochaete, with little doubt, represents
the incipient, plagio-orthotropic crust or cushion which finds its best expression
among the Chaetophorales, according to Fritsch (1942, p. 401), in Pseudo-
pringsheimia.
The genus has been reported to have only a single pyrenoid in each cell,
except by Hylmo (1916, p. 29) who found two in the longer cells; the present
examples of 0. lentiformis have frequently 2-3 pyrenoids to a cell.
CHAETOSPHAERIDIACEAE
Diplochaete solitaria Collins. This epiphytic alga was found at Woods Hole,
adhering to a filament of Polysiphonia Harveyi Bail. v. Olneyi (Harv.) Collins
collected in the wash at Nobska Point, on September 10, 1942. In cell measure-
ments, thickness of cell wall and characters of the solid setae, the specimen falls
completely within the description given by Collins. A pyrenoid was not seen;
its presence was doubted when the alga was first described. Only two individuals
were seen in the present instance and the alga appears to be rare.
Diplochaete remains a monotypic genus as when originally described, since
Polychaetophora W. et G. S. West, which was united with Diplochaete by
Collins (1909, p. 278), has been reestablished, and the genus Oligochaetophora
created for P. simplex G. S. West, a unicellular epiphytic form like D. solitaria
but found in fresh water (West, 1911, pp. 88-89).
Distribution: West Indies (Jamaica, on Laurencia obtusa); Massachusetts as
above.
Collins, 1901, p. 242; 1909, p. 277-78, figure 99; Wille, 1909, p. 103; Printz,
1927, p. 231, figure 178.
LITERATURE CITED
BATTERS, E. A. L., 1902. A catalogue of the British marine algae. Jour. Bot., 40: 1-107,
Supplement.
BORGESEN, F., 1915-20. Marine Algae of the Danish West Indies, II. Dansk Bot. Arkiv, 3:
1-504.
BORNET, E., AND C. FLAHAULT, 1888. Note sur deux nouveaux genres d'algues perforantes.
Jour, de Bot., 2: 161-165.
BORNET, E., AND C. FLAHAULT, 1889. Sur quelques plantes vivant dans le test calcaire^^de^
mollusques. Bull. Soc. Bot. France, 36: 147-174.
COLLINS, F. S., 1901. The algae of Jamaica. Proc. Amer. Acad. Arts and Sci., 37 (9): 231-270.
264 FRANCESCA THIVY
COLLINS, F. S., 1909. The green algae of North America. Tufts Coll. Stud., 2 (3): 79-480.
FELDMANN, J., 1937. Les algues marines de la cote des Alberes II, Chlorophyceae. Rev. Algol.,
9 (3-4): 173-241.
FRITSCH, F. E., 1935. The Structure and Reproduction of the Algae. Vol. I. Cambridge, Eng.
FRITSCH, F. E., 1942. Studies in the comparative morphology of the algae; I. Ami. Bot.,
'newser., 6 (23): 396-412.
HAMEL, G., 1930. Chlorophycees des cotes francaises. Rev. Algol., 5: 1-54.
HANSGIRG, A., 1892. Vorlaufige Bermerkungen iiber die Algengattungen Ochlochaete Crn. und
Phaeophila Hauck. Osterr. Bot. Zeitschr., 42 (6): 199-201.
HAZEN, T. E., 1902. The Ulothricaceae and Chaetophoraceae of the United States. Mem.
Torr. Bot. Club, 11: 135-250.
HEERING, W., 1914. Chlorophyceae III, in Pascher, A., Siisswasserflora Deutschlands, Oster-
reichs und der Schweiz, 6: I-IV, 1-250.
HUBER, M. J., 1892a. Observations sur la valeur morphologique et histologique des poils et
des soies dans les Chaetophorees. Jour, de Bot., 6: 321-41.
HUBER, M. J., 1892b. Contributions a la connaissance des Chaetophorees epiphytes et endo-
phytes et de leur affinites. Ann. Sci. Nat., Bot., VII, 16: 265-359.
HYLMO, D. E., 1916. Studien uber die marinen Grunalgen der Gegend von Malmo. Arkiv for
Bot., 14 (15): 1-55.
KLEBAHN, H., 1892. Chaetosphaeridium Pringsheimii, novum genus et nova species algarum
Chlorophycearum aquae dulcis. Jahrb. wiss. Bot., 24: 268-282.
KYLIN, H., 1935. Uber einige kalkborhende Chlorophyceen. Kungl. Fysiogr. Sallsk. i Lund,
Forhandl., 5 (19): 186-204.
LAKOWITZ, K., 1929. Die Algenflora der gesamten Ostsee. 747 pp. Danzig.
MIGULA, W., 1907. Algen. In Thome, Flora von Deutschland, Osterreich und der Schweiz,
VIB, 2 (IB): 513-917.
MOBIUS, M., 1892. Morphologic der haarartigen Organe bei den Algen. Biol. CentralbL,
12 (4): 97-108.
NADSON, G. A., 1927. Die kalkbohrende Algen des Schwarzen Meeres. Arch. Russ. Protistol.,
6: 147-53.
NEWTON, L. B., 1931. A Handbook of the British Seaweeds. British Mus. Nat. Hist. 478 pp.
London.
OLTMANNS, F., 1922. Morphologic und Biologic der Algen I: 1-459. Jena.
PRATT, H. S., 1935. A Manual of the Common Invertebrate Animals exclusive of Insects.
XVIII + 854 pp. Philadelphia.
PRINTZ, H., 1927. Chlorophyceae (nebst Conjugatae, Heterocontae und Charophyta). In
Engler, A., and K. Prantl, Die natiirl. Pflanzenfam., 2. Aufl., 3: 1-463.
REINKE, J., 1889. Algenflora der westlichen Ostsee deutschen Anteils. VI. Bericht der Komm.
zur wissensch. Unters. der deutsch. Meere. 101 pp. Berlin.
SCHUSSNIG, B., 1938. Vergleichende Morphologic der niederen Pflanzen I: I-VIII, 1-382.
Berlin.
TAYLOR, W. R., 1937. Marine Algae of the northeastern coast of North America. IX + 427
pp. Ann Arbor.
TAYLOR, W. R., 1942. Caribbean Marine Algae of the Allan Hancock Expedition, 1939. Univ.
South. California Publ. Allan Hancock Atlantic Exped., Report 2: 1-193.
THIVY, F., 1942. A new species of Ectochaete (Huber) Wille from Woods Hole, Massachusetts.
Biol. Bull., 83: 97-110.
WEST, G. S., 1904. British Freshwater Algae. XV + 372 pp. Cambridge, Eng.
WEST, G. S., 1911. Algological Notes. Jour. Bot., 49: 82-89.
WILLE, N., 1909. Conjugatae und Chlorophyceae in Die natiirl. Pflanzenfam., Nachtr. z. I, 2:
1-284.
POLARIZATION, KINETOCHORE MOVEMENTS, AND BIVALENT
STRUCTURE IN THE MEIOSIS OF MALE MANTIDS
SALLY HUGHES-SCHRADER
(Department of Zoology, Columbia University)
INTRODUCTION
The pioneer studies of mantid cytology of Giardina (1897), Oguma (1921)
and King (1931) were concerned primarily in establishing the chromosome
complement and the existence of the XiX2Y, o\ and X!XiX2X2, 9 , sex chromo-
some mechanism. The meiotic bivalents were found to conform to the usual
orthopteran types and few or no data were given on the prophase behavior of
any except the sex chromosomes. The later papers of Williams (1938) and
Erazi (1940), while in general supporting the earlier conclusions,- are based on
inadequate material and analysis. Erazi's report of an XY or XX sex chromo-
some complement in the male of Empusa pennicornis should be checked on more
extensive material.
The recent studies of White (1938, 1941) have resulted not only in his beautiful
analysis of the compound sex chromosome complex, and the discovery of an XO cf ,
XX 9 sex chromosome mechanism in many species, but have also disclosed in
the meiosis of male mantids three other problems of major cytological interest.
First is the nature of the bouquet stage. The occurrence of two separate polar-
izations of the chromosomes at different periods of the prophase, — the second of
which takes place at pachytene — offers an exceptionally favorable opportunity
for an analysis of bouquet formation. The second problem is posed by the
complicated series of kinetochore movements. In most mantid species the
spindle forms in late pachytene, whereupon homologous kinetochores move
suddenly apart toward opposite poles forcibly stretching open the bivalents in
the developing spindle. This unique movement is followed by the re-approach
of the homologous kinetochores as the chromosomes again contract; only then
do the bivalents move to their final position in the metaphase plate, following
which the real anaphasic movement is initiated. The third problem — the
relation of chiasmata to bivalent structure — is presented by the variation among
different species in the form of the bivalents during the stretching process and
at first metaphase. In Callimantis, where the stretch phenomenon is absent,
the bivalents retain the parallel association of their homologous chromosomes,
except for a localized separation at the kinetochore region, until anaphase. The
cytological evidence for the complete absence of visible chiasmata at all stages
in these bivalents is unequivocally clear (White, 1938; Hughes-Schrader, 1943).
In all the other species investigated by White (1941) the pre-metaphase stretching
of the bivalents discloses terminal connections between their homologous chromo-
somes suggestive of previous chiasma formation.
Obviously the amazing range of chromosome and more specifically kinetochore
movement, and in type of bivalent structure indicated above afford data which
265
266 SALLY HUGHES-SCHRADER
bear significantly on many problems of the mitotic mechanism. Until an
experimental analysis becomes possible, the comparative study of these phe-
nomena in many related species offers the best approach to the problems they
pose. The present report covers five species, in one of which are found two
distinct types of prophase behavior correlated with different geographic distribu-
tion. The cytological data are presented separately for each species,— followed
by a comparative study of the problems outlined above as clarified by the varia-
tions presented in the different species.
MATERIAL AND METHODS
Males of the following four species were collected on Barro Colorado Island,
Panama Canal Zone, during December of 1939 and 1940: Liturgousa annulipes
Serv., — six pre-adult nymphs, two adults; Stagmomantis Carolina Johann, — -two
adults; Angela guianensis Rehn, — one adult; and Choeradodis rhombicollis
Latreille, — two adults. Stagmomantis Carolina was also collected in the region
of Onancock, Virginia, in July and August 1941 and 1942; fifteen pre-adult and
two adult males were used in the present study. Also from Onancock is the
material of Paratenodera sinensis Saus., — comprising seven pre-adult males.
My experience confirms White's (1941) report that the pre-adult male offers the
most extensive range of stages in spermatogenesis. It is a pleasure to thank
Dr. James A. G. Rehn of the Academy of Natural Sciences of Philadelphia for
the identification of all of the specimens, with the exception of the Paratenodera.
The fixatives of Sanfelice, and Bouin as modified by Allen and Bauer, were
used exclusively. Material was sectioned at from 8 to 12 micra and variously
stained in iron haematoxylin, Smith's modification of Newton's gentian violet,
La Cour's chromic acid gentian violet, and Feulgen. Counterstaining with
erythrosin after gentian violet and with light green after Feulgen was useful for
spindle and nuclear membrane differentiation.
CYTOLOGICAL DATA
The three main problems — (1) the second polarization or bouquet stage,
(2) kinetochore movements, and (3) bivalent structure and chiasmata, will be
considered separately under the different species. Early prophase, prior to
pachytene, is remarkably uniform in all and a few words here will apply to all
the species studied. Leptotene and zygotene stages fix and stain poorly, and
no detailed analysis of them has been attempted. The major features of chromo-
some behavior can, however, be established. Leptotene is characterized by a
typical bouquet formation in which the ends of all the autosomes are aggregated
on a restricted region of the nuclear membrane immediately underlying the
division center, — while the bodies of the chromosomes extend through the nucleus
in loops. The sex chromosomes are also polarized. In Liturgousa and Angela
the single X undergoes a typical conflexion and its closely appressed ends are
aggregated with those of the autosomes. The behavior of the multiple sex
chromosomes of Stagmomantis, Choeradodis, and Paratenodera is less open to
analysis, but in Paratenodera at least it can be shown that the ends of all three
sex chromosomes are involved in the polarization. The bouquet formation
persists through zygotene and terminates at different times of the prophase in
SOME PROBLEMS OF MANTID MEIOSIS 267
the different species, but usually during early pachytene. By this time the
division center is no longer visibly differentiated from the general cytoplasm.
Stagmomantis Carolina from Virginia
The diploid complement of the male of this species is 27, — -12 pairs of auto-
somes plus Xi, X2, and Y. The chromosomes are figured and described by
King (1931); I shall therefore confine my account to those aspects of the meiotic
prophase bearing on the problems outlined.
/. Second polarization
With the cessation of polarization in early pachytene the chromosome ends
assume a random distribution in the nucleus, and a completely unoriented stage
ensues (Fig. 1). By mid pachytene the division centers again become visible as
asters form around them. Thereupon the chromosome ends once more orient
actively and aggregate on the nuclear membrane underlying the centers. For
the second time, therefore, in this prophase, a bouquet formation is brought about.
Two division centers are now involved, however, and this results in variation in
the pattern of polarization. Both ends of a bivalent may move to the same
pole, or one end may go to each pole stretching the body of the bivalent between
them. A double bouquet results — -with the ends of the bivalents variously
distributed at the two centers (Figs. 2 and 3). The centers are usually already
on opposite sides of the nucleus when they first become visible, but occasionally
the timing varies and the two poles may lie no more than 90° apart. In such
cases, as the centers continue to move toward opposite sides of the nucleus, the
grouped chromosome ends follow them on the inside of the membrane, for by
late pachytene the two bouquets are invariably some 180° apart. [This pre-
cocious activity of the centers and consequent shifting of the aggregated chromo-
some ends is more common in the Barro Colorado form of Stagmomantis and is
illustrated in Figures 12 and 13.] Such a sliding movement of the chromosome
ends along the membrane suggests that no real fusion or firm cementing of the
two is involved, — although after appropriate staining it is possible to demonstrate
that the terminal chromomeres of each bivalent are closely appressed against
the nuclear membrane (Fig. 4). The attenuation of the ends of the bivalents
might suggest that they are under tension, — but since it is equally apparent in
bivalents looped at one pole as in those stretched between two, this impression
is misleading. The apparent attenuation more probably stems from the fact
that as the gyres of the chromosomes increase in diameter and decrease in number
—progressively from the kinetochore distally — the ends are the last to be affected.
//. Kinetochore movements
a. Kinetochore separation, spindle formation, and stretch.
The nuclear membrane disappears early, while the nucleus is still in a late
pachytene stage. The spindle forms immediately, filling the entire nuclear
area, and undergoes a rapid elongation. Coincidently the bivalents are released
from their polarized condition. Thereupon their ends appear flaccid and evince
no further specific movement.
268
SALLY HUGHES-SCHRADER
The two kinetochores of each bivalent now separate sharply and orient toward
opposite poles (Fig. 5). This movement occurs while the bivalents are scattered
throughout the nuclear area, their position reflecting the grouping of the preceding
polarization (Figs. 5, 6, and 7). Bivalents lying close to one pole (upper right,
Fig. 6) may show as extreme an initial separation of kinetochores as those in
the equator.
(All drawings made with camera lucida at table level with Zeiss 2 mm., n.a. 1.3, obj. and
20 X oc.; enlarged with pantograph. Magnification as reproduced 2700 X.)
FIGURES 1 TO 4. Stagmomantis Carolina, Virginia.
FIGURE 1. Non-polarized early pachytene; upper level only drawn; ends distributed at
random. Feulgen.
FIGURE 2. Second polarization, in mid-pachytene. Feulgen; intact membrane and division
centers at poles unstained.
FIGURE 3. Later stage in second polarization. Feulgen.
FIGURE 4. Detail of same after gentian violet and erythrosin; terminal chromomeres applied
to membrane under center.
SOME PROBLEMS OF MANTID MEIOSIS
269
m '/•&$,
{$? "" "*"" r ~
FIGURES 5 TO 8. Stagmoniantis Carolina, Virginia.
FIGURE 5. Early stretch; membrane gone, upper bivalents still polarized, other bivalents
opening as kinetochores orient to poles. Feulgen.
FIGURES 6 AND 7. Early stretch; asynchrony of bivalents; secondary loci of separation
between homologues; spindle elongation complete. Feulgen.
FIGURE 8. Mid-stretch; some bivalents not yet opened; open cross in several bivalents
here and in Figure_9. ^_ Feulgen,
270 SALLY HUGHES-SCHRADER
The events just described — break down of nuclear membrane, formation and
elongation of spindle, separation and polar orientation of homologous kinetochores
—take place with great rapidity as is shown by the rarity of these stages compared
with those which precede and follow them. Other evidence supports this.
Thus, cells in which the nuclear membrane has but just collapsed, as shown by
the persistent polarization of some of the bivalents (top, Fig. 5), already show
continuous spindle fibers between the centers, and a marked increase in inter-
center distance over immediately preceding stages with membrane intact.
Again, in stages such as Figure 6, whose closeness to Figure 5 is attested by
evidence in the distribution of the chromosomes of their previous polarization,
the spindle has already attained almost its maximum length. Indeed, the
elongation of the spindle is always completed before all of the bivalents have
been stretched open by the poleward movement of their kinetochores. [Meas-
urements of spindle length at these and later stages are given in Table 1; a
consideration of the role of spindle elongation in the kinetochore movement is
reserved for the discussion — in comparison with data from the other species
studied.]
The movement of homologous kinetochores toward opposite poles continues
—often to an extreme degree. Not infrequently two thirds or more of the total
spindle length may be traversed by the separating kinetochores of a given biva-
lent. In the process, the homologous chromosomes of each bivalent are stretched
and pulled apart, retaining only terminal or subterminal connections in one or
both arms (Figs. 8 and 9). The resulting attenuation of the stretched chromo-
somes is extreme, often appearing to approach the breaking point (note especially
the middle bivalent of Figure 8). A pronounced asynchrony characterizes the
stretching process among the different bivalents. In its early stages, as pointed
out above, there seems no correlation between position in the spindle and time
and degree of stretch (Fig. 6) ; later stages however show the most extreme
stretch in bivalents equatorially placed on the spindle, while those nearer the
poles are belated in opening (Figs. 8 and 9). Eventually all the bivalents are
stretched open, — but the asynchrony is so great that the stretching process
actually overlaps the recontraction of the bivalents which follows it.
Due to the shortness of the pairing segments in the sex chromosomes, their
kinetochores are never so close together as those of the autosomal bivalents.
Furthermore, the chromosomes of the sex trivalent, unlike those of autosomal
bivalents, begin to separate and have already assumed an end to end alignment
(Xt~Y-X2) before the stretching process is initiated. (This point is difficult of
demonstration in Stagmomantis but is clearly evident in Paratenodera and
Choeradodis.) A$ the spindle forms in the nuclear area the kinetochores of the
sex trivalent move toward the poles. Their movement, unlike that of the
bivalents, is a random one, — not determined by repulsion between homologous
kinetochores. Thus one X and the Y may move toward one pole and the other
X toward the opposite pole; both Xs may go toward one and the Y toward the
other pole; or, one X may pass toward each pole while the Y is stretched between
them (Figs. 8, 9, and 10). Occasionally a trivalent shows all three kinetochores
near one pole, — but it is impossible to distinguish these positively from trivalents
which have not yet oriented. Apparently the direction of kinetochore movement
is toward the nearer pole, determined by the chance position of the chain of three
chromosomes at the time of the formation of the spindle.
SOME PROBLEMS OF MANTID MEIOSIS
271
b. Re-approach of homologous kinetochores.
The extreme stretching of the meiotic chromosomes is followed by their
gradual re-contraction and the re-approach of their widely separated kinetochores.
This movement seems to be brought about by the resumption of the normal
coiling of late prophase, previously interrupted by the stretching process. A
slight but consistent decrease in spindle length during the assumption of the
compact form of final metaphase (measurements in Table I) undoubtedly expe-
TABLE I
Kinetoclwre movement and spindle elongation
Measurements in ocular micrometer units; each figure is the average of some ten measure-
ments.
Inter-center Distance
Inter-kinetochore Distance
Pre-
Early
Late
Com-
Spindle
Pre-
Maxi-
Com-
Kineto-
Kineto-
stretch:
stretch:
stretch :
pact
elon-
stretch;
mum
pact
chore
chore
Species
mem-
brane
no
mem-
plate
form-
meta-
phase:
gation
during
mem-
brane
stretch;
(in
meta-
phase;
sepa-
ration
sepa-
ration
intact '
brane,
ing
plate
stretch
intact1;
longest
(in
during
at
no
com-
(av.
rod
longest
stretch
meta-
plate
pleted
max.
biva-
rod
phase
sep.)
lent)
biva-
lent)
Stagmomantis
Carolina, Va.
14.4
26.3
25.9
23.0
11.9
0.0
13.0
7.0
13.0
6.0
Stagmomantis
Carolina, B. C. Is.
19.0
24.2
24.1
22.7
5.2
2.0
10.5
7.2
8.5
3.3
Paratenodera sinensis
16.8
24.0
23.1
21.6
7.2
0.0
9.2
6.6
9.2
2.6
Liturgousa annulipes
11.5
24.5
23.9
24.0
13.0
2.1
10.6
9.2
8.5
1.3
Choeradodis
rhombicollis
26.3
28.7
27.6
26.6
2.4
2.0
7.1
6.1
5.1
1.0
1 Except in Liturgousa; here the inter-center distance recorded is the maximum attained
before the orientation of the kinetochores to the centers but after the breakdown of the nuclear
membrane.
dites the process. The outline of the chromosomes becomes smoother and their
staining capacity greater as contraction proceeds, suggesting the deposition of
matrical material during this time. This feature, together with the degree to
which the bivalent has been opened, permits one to distinguish between those
bivalents in process of stretching and those undergoing the ensuing contraction.
The precise time at which half spindle components form between the kinetochores
and the centers is uncertain, clue to the difficulty of differentiating them from
the mass of fine fibrils making up the continuous spindle. However, at the
period of maximum stretching many bivalents show unmistakable half spindle
components. The re-contraction of the stretched chromosomes, and the re-
approach of the homologous kinetochores, is thus effected despite any pull or
resistance that may be offered by the half spindle components.
The extent of these two opposite movements of the kinetochores, — first the
violent movement of homologous kinetochores toward opposite poles, second
their gradual re-approach to assume the position characteristic of metaphase,—
is shown by measurements of the distance between the kinetochores of a given
bivalent at the different stages. In the longest rod-shaped bivalent at the period
272
SALLY HUGHES-SCHRADER
of maximum stretch the distance between opposing kinetochores is 13 ocular
micrometer units; at metaphase it is 7 units (each figure is the average of measure-
ments in 10 nuclei). Since there occurs almost no separation of kinetochores
prior to spindle formation in the Virginia Stagmomantis, the first figure gives
the relative distance traveled by the kinetochores in their pre-metaphase poleward
movement. In their re-approach the kinetochores retrace nearly half this
distance. I would emphasize again the asynchrony of the different bivalents in
these two movements; the stretching of laggard bivalents continues pari passu
with the contraction of those which were first stretched open. Thus the kineto-
chores of one bivalent may be moving apart, toward opposite poles, while
those of others are re-approaching each other.
c. Formation of metaphase plate
While the contraction of the bivalents and the re-approach of their kineto-
chores is under way, a new movement of the chromosomes is initiated. From a
dispersed distribution through the whole spindle, the bivalents gradually shift
FIGURES 9 AND 10. Stagmomantis Carolina, Virginia.
FIGURE 9. Late stretch; one bivalent still unopened; movement to equator started; XiX2Y
in reorientation. Feulgen.
FIGURE 10. Early stage in metaphase orientation; XiX2Y mal-oriented. Feulgen.
into the equatorial region (Figs. 9 and 10). Each bivalent retains its bipolar
orientation during this movement and moves as a whole toward the equator.
The asynchrony noted in the two preceding movements is maintained in this
also: movement toward the equator proceeds concomitantly with the re-con-
traction of the bivalents, and may even overlap the stretching of the most
belated of them. The spacing of the chromosomes at the equator is at first
wide and open — but as the maximum degree of contraction is reached they move
in and form a fairly closely spaced metaphase plate. The accuracy of the
SOME PROBLEMS OF MANTID MEIOSIS
seriation of stages in these movements of late prophase is attested by the fact
that early anaphases always show the chromosomes in the compact form and
closely spaced arrangement of the final metaphase.
Perhaps the most baffling feature of the metaphase orientation lies in the
movements of the sex trivalent. We have seen that it assumes, at random,
a variety of orientations during the stretching process. If the two Xs have
moved to one pole and the Y to the other, the movement to the equator proceeds
as in the bivalents simply by contraction of the chromosomes and a shifting of
the whole configuration, with its orientation unaltered, into position in the
metaphase plate. But if any other orientation be assumed during the stretch—
if one X goes to each pole with the Y stretched between them, or if one X and
Y pass to one pole while the other X goes to the opposite pole — a re-orientation
ensues. One or more kinetochores shift their position and move through the
spindle so as to bring the two Xs opposite to the Y on either side of the equator.
Thus one kinetochore actually changes its orientation from one pole to the other
and moves from a position close to one center to the opposite side of the equator
—a maneuvre difficult indeed to visualize in terms of the mechanics of mitosis.
Variation in the timing of the stretching of the trivalent and of its re-orientation,
relative to the activities of the bivalents, makes it difficult to seriate its move-
ments. But the trivalent shown in Figure 9 is probably undergoing re-orienta-
tion; the X near the upper pole is still under tension and maintains the orientation
assumed in the stretch, while the lower X shows no tension and its kinetochore
is in process of shifting toward the upper pole. What happens to the half
spindle components during re-orientation would be of great interest, but I have
not been able to follow it. Half spindle fibers are occasionally clearly visible
during the stretch and are again well marked at metaphase; it is possible that
they are lost and reform anew during the re-orientation.
Although the details of the process of re-orientation thus cannot be followed
with certainty, — there can be no doubt that it actually takes place. Mai-
orientation is encountered in some 50 per cent of the nuclei during the period of
the stretch [56 cases were observed among 105 counted], — while at the final
metaphase it is extremely rare [three cases in 105]. Clearly, therefore, the great
majority of those trivalents mal-oriented during the stretch successfully re-orient
by final metaphase.
///. Bivalent structure and chiasmata
Throughout pachytene the homologous chromosomes of each bivalent retain
their close parallel association, with but a slight tendency to separate at the
kinetochore region (Figs. 2 and 3). Diplotene and diakinetic stages as ordinarily
recognized are absent: they are replaced by the forcible stretching open of the
bivalents as the spindle forms, and the two kinetochores of each bivalent move
toward opposite poles. As stretching proceeds it becomes evident that the
kinetochore loop is not always the only locus of separation between homologues
(note the two centrally placed bivalents in Figure 5). These openings, loops
or half loops, alternate with persistently paired regions which resist the opening
out process (Figs. 5 and 6). The paired segments may be terminal or inter-
stitial, in one arm or in both; they vary in number from one to a maximum of
three per bivalent. When first observable the openings between the paired
274 SALLY HUGHES-SCHRADER
regions appear to lie in the same plane, but as the tension in the kinetochore
loop increases they may assume alternating planes (Fig. 6, middle left bivalent).
The chromatids of each chromosome have not yet separated, and even the line
of demarcation between chromosomes cannot be followed in the closely paired
regions. It is thus impossible to determine whether or not chiasmata are present
in the persistently paired regions. At the stage of maximum stretch, however,
open cross formations are frequently encountered either at one end of a bivalent,
giving the rod-shaped configuration, or at both with a resulting ring configuration
(Fig. 8). In these bivalents it is clear that non-sister chromatids are associated
distal to the opening of the cross.
No open cross configurations have been found in the sex trivalent. There is
thus no evidence available as to whether or not the terminal adhesions of Xi, Y,
and Xo are of chiasmatal origin.
The final form of the bivalents at completed metaphase is fairly constant
(Fig. 11). The most frequent complement comprises three rings and nine rods;
FIGURE 11. Stagmomantis Carolina, Virginia. Bivalents and sex trivalent at metaphase.
Gentian violet.
rings vary in number from none to four per nucleus — rods show the corresponding
range of 12 to eight. The open cross may persist to metaphase in from one to
four bivalents, but the terminal connection is more commonly a swelling or
lump, sometimes bipartite.
Stagmomantis Carolina from Barro Colorado Island
Taxonomically indistinguishable, Stagmomantis Carolina males from Barro
Colorado Island and from Virginia are also identical in chromosome complement
as observed in spermatogonial and meiotic metaphases. Striking and constant
differences, however, characterize the meiotic prophase in the two types.
In the Barro Colorado material the pachytene polarization is initiated always
at a later stage in the development of the bivalents than in the Virginian, and is
of shorter duration (compare Figures 12 and 2). Moreover there is here no
constant correlation between time of polarization, degree of separation of the
centers, and the stage of bivalent development. Thus in Figure 13 the polariza-
tion centers are active while still relatively close together, but the bivalents are
in a more advanced stage than those of Figure 12 in which the centers are already
at opposite sides of the nucleus. The time of breakdown of the nuclear membrane
also varies relative to the degree of separation of the centers. When the centers
SOME PROBLEMS OF MANTID MEIOSIS
275
separate early their passage to opposite sides of the nucleus is accompanied by
a marked elongation of the whole nucleus (Figure 12 is typical) along the inter-
center axis. Thus when the spindle forms in the nuclear area on the collapse of
the membrane, the average inter-center distance is already considerably greater
than in the Virginia type. Some elongation of the spindle follows immediately
on its formation but the total length achieved is somewhat less and the average
amount of elongation considerably less than in the Virginia material (measure-
ments in Table I). The maximum separation of homologous kinetochores
12
I
t> J£jk ^*iy. . "• ut
FIGURES 12 AND 13. Stagmomantis Carolina, Barro Colorado.
FIGURE 12. Second polarization, late pachytene; early separation of homologous kineto-
chores. Haematoxylin.
FIGURE 13. Second polarization with centers till close together; early breakdown of mem-
brane; advanced stage of bivalent opening. Haematoxylin.
during the stretching process is definitely less in the Barro Colorado type; this
is probably dependent both on the more advanced stage of bivalent contraction
and the lesser spindle elongation.
A highly significant feature of the Barro Colorado type lies in the timing
of the separation of homologous kinetochores. In the Virginia form this occurs
simultaneously with the orientation of the two kinetochores of each bivalent to
opposite centers — -and only after membrane collapse and spindle formation. In
bivalents of the Barro Colorado material, the homologous chromosomes show a
marked localized separation at the region of the kinetochores while the nuclear
membrane is still intact (Fig. 12). By the time the membrane gives way the
separation of homologues, initially localized at the kinetochore region, has spread
276 SALLY HUGHES-SCHRADER
distally until in some cases the bivalent appears as a ring with only the terminal
regions of the chromosomes still parallelly associated (Fig. 13). It is clearly
evident in these stages (Figs. 12 and 13), that the plane of separation between
homologous kinetochores bears no relation to the future spindle axis. This fact,
together with the persistance of the nuclear membrane during the initial separa-
tion, demonstrates therefore that this first phase in the kinetochore movement is
independent of centers and spindle.
With the collapse of the membrane and formation of the spindle in the nuclear
area, the already widely separated kinetochores of each bivalent orient and move
toward opposite poles (Fig. 14). As in the Virginia type, this first phase in the
stretching process occurs while the chromosomes are scattered through the whole
spindle; Figure 15 is a particularly striking example with four bivalents, all
placed well above the equator, showing the movement of the kinetochores to
opposite poles. In this and succeeding stages however the stretch is always
most extreme in equatorially placed bivalents.
The asynchrony of the bivalents in the stretching process is equally marked
in both types (compare Figures 14 to 16 with 6 to 9). In both the maximum
spindle length is attained before all of the bivalents have completed the
stretching process. The structure of the bivalents as disclosed during the
stretch is also identical in the two types. Re-contraction of the chromosomes,
re-approach of homologous kinetochores, and the movement to the equatorial
plate proceed similarly. The basic difference between the two types thus lies in
the timing of spindle formation relative to the stage of bivalent development.
The more precocious spindle formation in the Virginia form superimposes the
initial separation of homologous kinetochores and their bipolar orientation.
With the delay in spindle formation in the Barro Colorado type the two processes
are seen to be distinct; the initial separation of kinetochores is not determined
by the centers nor the developing spindle.
Paratenodera sinensis
The chromosomes of this species have been studied and figured by King
(1931) and White (1941). The diploid number of the male is 27, 12 pairs of
autosomes plus Xi, X2 and Y.
/. Second polarization
Pachytene polarization in Paratenodera presents an interesting and significant
variation from the pattern observed in Stagmomantis. Usually polarization is
not marked until the bivalents have condensed into short thick rods. No
diplotene opening out, however, accompanies this prolonged period of contrac-
tion. The bivalents, scattered widely in the nucleus, evince some tendency
toward peripheral distribution; some loose collocation of ends may persist from
the leptotene zygotene bouquet, but no regular orientation is apparent. Asters
form while the centers are still fairly close together. Only then do the chromo-
somes become definitely polarized, forming two loose aggregations close to the
nuclear membrane and underlying the centers (Fig. 18). So compact are the
bivalents at this stage that it is impossible to say whether their ends only or the
whole mass is involved in the polarization. However the time at which the
SOME PROBLEMS OF MANTID MEIOSIS
277
16 "^1
FIGURES 14 TO 16. Stagmomantis Carolina, Barro Colorado.
FIGURE 14. Early stretch. Haematoxylin.
FIGURE 15. Mid stretch; bivalents still scattered; early stage in open cross in one bivalent.
Gentian violet.
FIGURE 16. Early stage in metaphase orientation ; mal-orientation of sex trivalent. Gentian
violet.
278
SALLY HUGHES-SCHRADER
17
FIGURES 17 AND 18. Paratenodera sinensis.
FIGURE 17. Early second polarization with chromosome ends polarized; sex trivalent in
outline. Gentian violet.
FIGURE 18. Typical second polarization. Haematoxylin.
SOME PROBLEMS OF MAXTID MEIOSIS 279
centers become active varies in relation to the stage of chromosome contrac-
tion. In a small percentage of the cells the asters form while the* bivalents
are still fairly long, and it is then evident that the ends of the chromosomes are
specifically involved (Figure 17) just as in Stagmomantis. The Paratenodera
material thus affords a series of transitional phases linking bouquet formation
with the type of late prophase polarization found in Anisolabis (Schrader, 1941a)
in which a specific activity of chromosome ends is not evident.
// and III. Kinetochore movements and bivalent structure
Events subsequent to the cessation of pachytene polarization follow the same
general course as in Stagmomantis. Like the Virginia type of the latter, the
20
21
FIGURES 19 TO 22. Liturgousa annulipes.
FIGURE 19. Spermatogonial metaphase. Gentian violet.
FIGURE 20. First meiotic metaphase. Univalent X off plate at right, viewed from open
end of Y. Gentian violet.
FIGURE 21. Second meiotic metaphase — 11 autosomes and X. Gentian violet.
FIGURE 22. Second meiotic metaphase — 11 autosomes and no X. Gentian violet.
bivalents show no marked separation of kinetochores prior to the collapse of the
membrane. Formation and elongation of the spindle coincides with the initial
opening out of the bivalents but the stretching process continues after spindle
growth is complete. Degree of spindle elongation and intensity of stretch are
intermediate between the Virginia and Barro Colorado types of Stagmomantis
(measurements in Table I). Re-contraction of bivalents, re-approach of kineto-
chores, and movement to the equator show no significant differences from the
280 SALLY HUGHES-SCHRADER
conditions in Stagmomantis. The structure of the bivalents also corresponds
closely in* the two genera.
Liturgousa annulipes
The chromosomes of this species have not previously been recorded. The
diploid number of the male as seen in spermatogonial metaphase is 23, com-
prising 11 pairs of autosomes and a single X (Fig. 19). The kinetochore is
approximately median in six pairs of autosomes, subterminal in five, and sub-
median in the X (Figs. 19 and 27). The X passes undivided to one pole at the
first division and divides at the second. Second metaphase shows the expected
chromosome sets of 12 and 11 (Figs. 21 and 22).
/. Second polarization
The pachytene polarization of the bivalents is very slight (Fig. 23). Many
cells show none at all, and the maximum observed involves the orientation of one
or both ends of at most three to five bivalents — probably those which happened
to lie near the center when it became active. The time of polarization coincides,
as in all the other species studied, with the first formation of astral rays. In
Liturgousa, however, the latter make their appearance while the centers are still
either undivided or so close together that only a single center of polarization is
produced.
II. Kinetochore movements
a. Kinetochore separation, spindle formation and stretch.
As in the Barro Colorado Stagmomantis, the initial separation of homologous
kinetochores is independent of division centers and spindle. This is shown, as
in the former case, by the marked "repulsion" of kinetochores which occurs,
in many bivalents, from mid-pachytene on (Figs. 23 and 24). Since the nuclear
membrane is still intact, the future spindle axis not yet established, and the
plane of kinetochore separation is random, any influence of the achromatic
figure on the kinetochore movement is excluded.
The nuclear membrane breaks down while the centers are still close together
(Fig. 25). The subsequent reaction of kinetochores to centers is slow in compari-
son with the other species, but gradually all the bivalents become oriented with
one kinetochore moving toward each pole. Meanwhile the spindle forms and
elongates. Due to the late separation of the centers spindle elongation after
membrane collapse is greater than in any of the other species (Table I). But,
as in the other forms, its maximum length is attained before the stretching of
the bivalents is completed. A late stage in the stretching process, with one
bivalent not yet completely opened, is shown in Figure 26.
FIGURES 23 TO 26. Liturgousa annulipes.
FIGURE 23. Second polarization; slight orientation of chromosome ends near center; division
centers not yet separated. Haematoxylin.
FIGURE 24. Same stage — no polarization; kinetochores separating in various planes.
Haematoxylin.
FIGURE 25. Slightly later; membrane gone; centers separating; delayed orientation of
kinetochores. Haematoxylin.
FIGURE 26. Late stretch; metaphase orientation started. Gentian violet.
SOME PROBLEMS OF MANTID MEIOSIS
281
24
25
26
FIGURES 23 TO 26.
SALLY HUGHES-SCHRADER
b. Re-approach of homologous kinetochores; metaphase plate formation.
The re-contraction of the bivalents, re-approach of the homologous kineto-
chores, and the gradual movement toward the equatorial region proceed precisely
as in Stagmomantis.
///. Bivalent structure and chiasmata
In spite of the markedly early separation of the kinetochores and the conse-
quent opening out of the bivalents before spindle formation, the chromatid
structure of the bivalents is not analyzable at this stage.. The separation of
homologous chromosomes, at first localized in the kinetochore region, continues
until a large kinetochore loop is formed, or, in some bivalents, the homologues
become completely disjoined except for one short paired segment. Sister
chromatids remain closely associated and cannot be traced through the per-
sistently paired regions. That the separation of homologous chromosomes is
not solely due to kinetochore movement is shown by the presence in some bi-
valents of a second locus of opening out in addition to the kinetochore loop or
half loop. This is apparent in the separation of the ends of the horizontally
placed bivalent at the mid left in Figure 24, and in all bivalents of Figure 25.
After spindle formation and during the stretching process the open cross formation
i 23 456789 10 (IX
FIGURE 27. Liturgousa annulipes. Eleven bivalents and X at completed metaphase.
Gentian violet.
is encountered in several bivalents (Figures 26 and 28, a and b). The frequency
of open crosses is 11.6 per cent (in 249 counted) during the stretch, and is reduced
to 5.4 per cent (in 351) by final metaphase.
The open cross configuration is usually accepted as evidence of a chiasma in
process of resolution by the rotation of the arms of the bivalent. We might
then assume, as White (1941) has suggested, that the terminal connections
between the homologous chromosomes of the bivalents at metaphase are of
chiasmatal origin. If a chiasma were present in each arm, two terminal con-
nections would be formed — giving a ring bivalent at metaphase; if only one arm
contained a chiasma, a single terminal connection and a rod bivalent would
result. The behavior of two of the Liturgousa bivalents does indeed support
such an argument. Easily identifiable at metaphase is the small bivalent number
2 of Figure 27 in which a terminal connection between the short arms always
persists throughout the stretch, while the long arms are always free. The large
SOME PROBLEMS OF MANTID MEIOSIS
bivalent number 3 of Figure 27 has arms of similar proportions, but in its case
the long arms as well as the short ones are invariably connected. Moreover, in
this bivalent the open cross formation may occasionally be observed in the long
arms during the stretch (Fig. 28, c). Since the short arms only are subjected to
stress in both bivalents, it seems reasonable to assume that the difference in
behavior of the long arms is associated with the presence or absence of chiasmata.
FIGURE 28. Litiirgousa annulipes. a and b, open cross formation in rod bivalents; c, open
cross in bivalent no. 3; d, bivalent no. 1 during stretch. Gentian violet.
On this assumption, if no chiasma is present, the homologues separate completely
and early as in the long arm of number 2, while the presence of a chiasma results
in a persistent terminal connection as in the long arm of number 3.
It does not follow, however, that chiasmata are the sole and essential factor
in maintaining the association of homologues. One is not justified in generalizing
from the behavior of these two bivalents. Some bivalents never show the open
cross or any other evidence of chiasmata; their homologues separate during the
stretch with sister chromatids persistently associated; the connection between
chromosome ends in these cases carries no necessary implication of previous
chiasmata. Even stronger evidence is afforded by the behavior of bivalent
number 1 (Figure 27). It is readily identifiable at metaphase because, alone of
all the bivalents, its constituent chromosomes are in parallel association except
at the kinetochore. It thus, except for a somewhat closer association of chroma-
tids, strikingly resembles the bivalents of Callimantis (White, 1941; Hughes-
Schrader, 1943) in which the absence of chiasmata in late prophase and metaphase
is definitely demonstrable. But unlike Callimantis, in Liturgousa this bivalent
is subjected to the same stretching process which reveals in the other bivalents
of the set those open crosses and terminal connections suggestive of chiasmata.
It is, therefore, evidence of a real difference in the factors determining bivalent
structure to find that the homologous chromosomes of bivalent 1 separate
smoothly and progressively during the stretch, disclosing no evidence of chias-
mata, and making no terminal knots or adhesions. The paired region at the end
of each arm decreases steadily in extent as the bivalent is stretched, but retains
some parallel orientation of its chromosomes as long as it can be followed (Fig.
28, d). Re-contraction of bivalent 1 after the stretch brings the separated
284 SALLY HUGHES-SCHRADER
chromosomes back into parallel association throughout their length, except at
the kinetochore. If the absence of chiasmata permits the chromosomes of the
long arm of bivalent 2 to separate early and completely, why do the arms of
number 1, apparently equally devoid of chiasmata, retain the parallel association
of their homologues? Conversely, the pairing force operative in number 1
31 33
FIGURES 29 TO 33. Choeradodis rhomb icollis.
FIGURE 29. Typical spermatogonial metaphase. Gentian violet and erythrosin.
FIGURE 30. Same, with maximum association of homologues. Gentian violet and ery-
throsin.
FIGURE 31. First meiotic metaphase; sex trivalent at upper center with Y at top focus and
2 Xs, stippled, at lower focus. Gentian violet.
FIGURE 32. Second meiotic metaphase, 14 autosomes plus 2 X. Gentian violet.
FIGURE 33. Same, with 14 autosomes plus Y. Gentian violet.
SOME PROBLEMS OF MANTID MEIOSIS
285
would seem to be absent in the long arm of number 2. Variation in the factors
determining both the association of homologous chromosomes and the form of
the metaphase configurations is thus clearly indicated for different bivalents
within a single complement. Further consideration of these data is given in
the discussion.
Choeradodis rhombicollis
The diploid number in the male of this species, as determined in spermatogonia,
is 31 — the highest number thus far found among mantids (Figs. 29 and 30).
Williams' (1938) count of 27 is based on a single adult fixed without dissection,
and his figures indicate that the material was inadequate to establish chromosome
number and behavior.
FIGURE 34. Choeradodis rhombicollis. Pseudo-diakinesis following diffuse late pachytene.
No polarization; bivalents peripheral; erythrosin stained coagulum about chromosomes. Gentian
violet and erythrosin.
The chromosome complement embraces 14 pairs of autosomes, four with
median, 10 with subterminal kinetochores, and 3 sex chromosomes, Xj. X2 and Y,
with submedian kinetochores. Homologous chromosomes tend to lie near one
another at spermatogonial metaphase ; Figure 30 shows the maximum association
observed, Figure 29 the more typical condition. The sex chromosomes are
positively heteropycnotic in spermatogonial prophase and telophase, and lag in
the anaphase movement.
Fourteen bivalents and a sex trivalent are formed at meiosis (Fig. 31). In
general structure and behavior the trivalent corresponds with those described
for other species. The proportions of the arms and the pairing relations at
286
SALLY HUGHES-SCHRADER
metaphase are shown in Figure 38. At the first meiotic division the 2 Xs pass
to one pole, the Y to the other; the second metaphase accordingly shows either
15 or 16 elements (Figs. 32 and 33).
/. Second polarization
Leptotene, zygotene, and early pachytene stages correspond closely to those
of the other species studied. In late pachytene however, the chromosomes
become diffuse in outline, are almost unstainable in gentian violet and haema-
toxylin, and give but a faint Feulgen reaction. When later they again become
stainable, the bivalents, already greatly shortened compared to the long threads
of early pachytene, are found peripherally distributed close to the nuclear mem-
FIGURE 35. Choeradodisrhombicollis. Second polarization; chromosomes loosely aggregated
near division centers. Haematoxylin.
brane (Fig. 34). Asters form about the centers, and these separate quickly to
opposite sides of the nucleus, which may elongate slightly along the inter-center
axis (Fig. 35); but the polarization is always slight and many do not reach the
polar regions. So late is the polarization relative to the stage of contraction of
the chromosomes that the latter appear to move as wholes. In a few nuclei,
however, polarization occurs while the chromosomes are still relatively long, and
then, as in Paratenodera, the ends alone orient to the center.
//. Kinetochore movements
On the breakdown of the nuclear membrane the spindle forms in the nuclear
area and quickly elongates. Due to the large size of the nucleus and the conse-
SOME PROBLEMS OF MANTID MEIOSIS
287
quent wide separation of the centers, the length of the spindle is considerably
greater than in any of the other species, — but the extent of its elongation is
relatively slight (comparative measurements in Table I). As in the other species,
spindle elongation is completed before the opening out of many of the bivalents.
Indeed the orientation and movement of homologous kinetochores toward
opposite poles takes place very gradually and with pronounced asynchrony in
this species. Figure 36 is typical; the spindle has completed its elongation; of
FIGURE 36. Choeradodis rhombicollis. Stretch: elongation of spindle completed; asynchrony
of bivalents in opening out. Gentian violet.
the bivalents some still retain the parallel association of their homologues except
at the kinetochores, while others have completed the stretching process. The
degree of stretching is very slight compared to the other species; the maximum
separation of kinetochores observed is less than a third of the length of the
spindle. The contraction of the bivalents to their final metaphase form is
similarly slight. The further movements of the chromosomes to the equator
and their orientation in the metaphase plate proceed as in the other species.
The behavior of the sex trivalent parallels in essentials that of Stagmomantis.
The early terminal alignment of the sex chromosomes, prior to the breakdown
of the nuclear membrane, is especially clear in Choeradodis.
///. Structure of bivalents
Unfortunately in Choeradodis, in which among all the species thus far studied
the closest approach to a regular diplotene-diakinetic opening out occurs, the
structure of the bivalents is least analysable, — due to their small size and the
diffuse condition of the chromatin in the critical stages. Certain significant
features can, however, be established.
288
SALLY HUGHES-SCHRADER
Already on emergence from the confused stage of late pachytene the bivalents
show a marked degree of separation between homologues (Fig. 34). This may
be uniform along their entire length, or variously accentuated in different regions.
If uniform, the homologues are clearly separated and indubitably devoid of
chiasmata (Fig. 37, a, after Feulgen, and e, gentian violet). [Occasionally a cross
section of such a bivalent shows all four chromatids equally spaced (Fig. 37, f),
* u
.-,*
* *.
-* »
f
h
FIGURE 37. Choeradodis rhomb icollis. Individual bivalents at pseudo-diakinesis. a-d,
Feulgen; e-h, gentian violet.
but such figures are rare and I have not observed chromatid separation in any
others.] Prometaphase bivalents of this type probably assume a Callimantis-like
form at metaphase as in bivalent 2 in Figure 38, or if more widely opened, produce
simple rings such as bivalents 1 and 3 in the same figure.
In other bivalents, homologues are moderately and uniformly separated at
one end, but flare widely apart at the other as though mutually repelling each
I 2 34 5 6 7 8 9 10 II 12 13 14 X
FIGURE 38. Choeradodis rliombicollis. Bivalents and sex trivalent at first metaphase.
Gentian violet.
other (Fig. 37, c). If at metaphase a terminal attraction operates between the
paired ends when the poleward movement of the kinetochores has brought them
into apposition, a rod-shaped bivalent, with no implication of previous chiasma,
will result.
SOME PROBLEMS OF MANTID MEIOSIS
289
A third type of association is shown in Figure 37, b, d, g, and h. Here the
persistently paired region, usually terminal or subterminal, is very short and the
hornologues separate widely throughout the rest of their length, with maximum
separation usually at the kinetochore region. Homologous chromosomes are in
%**
40
41
43
FIGURES 39 TO 43. Angela guianensis.
FIGURE 39. Stretch stage. Haematoxylin.
FIGURE 40. First meiotic metaphase; X lies off plate at low focus. Gentian violet.
FIGURES 41 AND 42. Second meiotic metaphases. Gentian violet.
FIGURE 43. Bivalents and X at compact metaphase. Gentian violet.
contact in the paired region, but since chromatids cannot be distinguished it is
impossible to tell whether or not a chiasma is present. The possibility that
chiasmata are involved in these contact points is strengthened by the occasional
occurrence of open cross formations in the later stages (Fig. no. 5). The fre-
quency of the open cross is low, — slightly less than 1 per cent at metaphase.
290 SALLY HUGHES-SCHRADER
Taken as a whole the Choeradodis data support the conclusion derived from
the Liturgousa bivalents: the factors determining the association of homologous
chromosomes in late prophase and the form of the bivalents at metaphase vary
in different bivalents of a single species. Additionally Choeradodis demonstrates
that in certain cases a pairing force independent of chiasmata is variously localized
along the bivalent.
Angela guianensis
My material of this species is limited to a single adult male, in which the
stages of spermatogenesis are incompletely represented. Its chromosomes have
not previously been described. The diploid number in the male is 19, deter-
mined from meiotic metaphases (Figs. 40 to 43). Six pairs of autosomes and
the univalent X have median — the others submedian kinetochores. At meta-
phase the bivalents assume the familiar rod or ring form; the number of rods
varies from 3 to 5, of rings from 4 to 7; the most common complement being 3
rods and 6 rings (Fig. 43).
Early prophase is typical. Pachytene polarization is represented by but few
nuclei but these indicate that it occurs relatively early as in the Virginia Stagmo-
mantis. The most striking feature of the prophase is the extreme delicacy of
the chromosomes at the time of stretching — a chromomeric structure is still
present during the period of the stretch (Figure 39). Spindle formation seems
to be more precocious than in any other species. The spindle is very compact,
nearly spherical in form, and the stretched chromosomes are curved in conformity
with it. During the stretching and subsequent re-contraction of the bivalents,
open cross configurations are frequent, — -nearly every nucleus in these stages
showing two or three. These are resolved and are succeeded by terminal con-
nections in final metaphase.
The low chromosome number, precocity of spindle formation, and frequency
of open cross formation suggest that this species would reward further study.
DISCUSSION
/. Polarization
The second polarization of chromosomes in the mantid prophase is of especial
significance in the analysis of the bouquet stage — so characteristic a feature of
meiosis in many animals, and to a lesser extent, plant species. The interpolation
of a non-polarized stage just prior to the second bouquet demonstrates that the
latter cannot be interpreted simply as a passive relic of previous telophase
orientation, but involves an active orientation of chromosomes. Furthermore,
the second bouquet occurs at a stage more open to analysis than is the leptotene.
It is thus possible to demonstrate the participation of three structural elements
of the cell in the process of polarization: (1) the orientation movements are
performed by the ends of the chromosomes; (2) the division center determines
the focus of aggregation ; and (3) the nuclear membrane is involved in polarization.
Evidence on all these points has long been available. Recognition has been
delayed due to a confusion of two distinct types of orientation: (1) the active
orientation of chromosomes to membrane and center so frequently encountered
in meiotic prophase, and (2) the superficially similar Rabl orientation — a passive
relic of previous telophase orientation — which has been demonstrated with
SOME PROBLEMS OF MANTID MEIOSIS 291
perhaps equal frequency in mitotic prophase. Confusion has arisen because in
meiotic division the first process is sometimes superimposed upon the second.
If the true leptotene bouquet is formed in cells which retain the pre-meiotic
telophase orientation of their chromosomes it may well be impossible to discrimi-
nate between two distinct but simultaneously acting factors — the Rabl or relic
orientation and the active orientation of bouquet formation. A third factor
may further complicate polarization; it lies in the mutual attraction, at certain
phases in the mitotic cycle, of heteropycnotic regions of the chromosomes. If
heteropycnosis is terminally localized in the chromosomes and the attraction
operates simultaneously with peripheral distribution and a polarizing activity of
the center, a bouquet formation will result (as, for example in Phrynotettix
(Wenrich, 1916), Stauroderus (Corey, 1933, 1938), and several species of Edessa
(Schrader, 1941b)). The numerous well established cases in which bouquets form
without heteropycnosis, as well as those in which heteropycnotic regions aggregate
without bouquet formation, show that two separate processes are involved.
The recent revival of the idea that the bouquet stage is conditioned by
previous telophasic orientation (Atwood, 1937; Hiraoka, 1941; Smith, 1942)
demands reconsideration of the evidence, even at the risk of repetition. The
basis for the idea lies primarily in the large body of evidence establishing the
relative immobility of chromosomes during the resting stage. The aggregation
of the kinetochores close to the division center in late anaphase brings the chromo-
some arms into a parallel or radiating arrangement. The closing in of the new
nuclear membrane might then tend to bring the ends together. Granted relative
immobility through the resting stage, an approximation of the bouquet arrange-
ment would thus be already determined by the preceding telophase orientation.
The evidence against this interpretation of the bouquet has been presented
by Schrader (1941b); I will summarize it here. First, telophasic reorganization
will tend to bring chromosome ends together only if the kinetochore is median
or nearly so. The formation of a typical leptotene bouquet in Phrynotettix
(Wenrich, 1916) whose chromosomes have effectively terminal kinetochores thus
shows that half of the chromosome ends have moved through the nucleus and
oriented at the pole opposite that approached at telophase. Again, the hy-
pothesis would demand a definite and fairly uniform chromosome length. In
Choeradodis, with marked size differences among its chromosomes, the leptotene
bouquet shows the kinetochore of the shortest element to be relatively close to
the pole at which the ends are aggregated, while in only the longest chromosomes
with median kinetochores do the latter retain their telophasic position. Third,
typical bouquets occur in several Hemiptera (Geitler, 1937; Schrader, 1941b)
although a kinetochore-center telophase aggregation cannot here — due to the
diffuse nature of the kinetochore — be a causal factor (see, however, some contrary
evidence in Ris, 1942). The converse of this argument is also applicable: many
forms with pronounced aggregation of kinetochores at the center in telophase
fail to show any bouquet stage in the succeeding meiotic prophase.
Again, we are confronted in many cases with an apparent shift in the spatial
relations of center and kinetochores between telophase and meiotic prophase.
Thus in Locusta (Mohr, 1916) the median kinetochores are aggregated near the
center in telophase but prophase shows them at the opposite side while the
chromosome ends are now grouped at the center. None of the suggestions
292 SALLY HUGHES-SCHRADER
seeking to reconcile these conditions independently of active chromosome orienta-
tion, such as a rotation of the nuclear contents through 180° (Janssens, 1924;
Geitler, 1934) or a migration of the center through a similar arc (Schreiner and
Schreiner, 1906; Gelei, 1921), meets the basic objection that polarization in the
bouquet involves a more precise orientation and pronounced focussing of ends
to a single circumscribed region than any telophasic orientation and reorganization
would entail.
The formation of a typical bouquet in late prophase, following a completely
non-polarized stage such as occurs in certain of the mantids, proves the meiotic
polarization to be a distinct process involving forces not operative in relic orien-
tation. The mantid evidence is valuable for its clarity rather than its novelty.
Similar evidence has been available since 1921 in Gelei's careful analysis of the
formation of the leptotene bouquet in Dendrocoelum oocytes. He was able to
count close to the total number of chromosome ends in the early pre-bouquet
leptotene nucleus, and found them distributed, peripherally, but at random
relative to the division center, throughout the nucleus.
Finally, the evidence just presented although sufficiently conclusive in itself,
is secondary to the basic fact now also well established that bouquet formation
involves a special activity of chromosome ends, center, and nuclear membrane.
The action of the chromosome ends in polarization is clearly evident in
bouquet formation in mantids, as in many other organisms. Gelei (1921) was
able to follow the movement of the knobbed ends of the Dendrocoelum chromo-
somes as they converged from a random distribution to aggregate in a plate-
like cluster on the nuclear membrane underlying the center. The same process
occurs in the formation of the second bouquet in certain mantid species.
Furthermore, the variation in timing of polarization relative to degree of chromo-
some contraction seen in Paratenodera provides transitional stages between
polarizations in which ends only are active, and those in which the whole compact
body of the chromosome seems to be affected. This suggests that in the latter
type, also, a special activity of ends may be involved, and adds to the growing
body of evidence bespeaking special functions in these structures.
Action of the center in determining the pole of the bouquet has also long been
recognized (Buchner, 1910; Ahrens, 1936, and others). It is strikingly evident
in those mantid nuclei in which a single or monopolar bouquet first forms at the
still undivided center and is then transformed into a double, bipolar, bouquet as
the daughter centers move to opposite sides of the nucleus accompanied each by
a group of chromosome ends on the inside of the membrane.
That the nuclear membrane takes a definite, if still undefined, part in polar-
ization is becoming increasingly clear. Schrader (1941a) has shown that it
plays more than a passive role in the movements of the centers as well as of the
chromosomes. In the mantids the close application of terminal chromomeres to
nuclear membrane, and the abrupt cessation of polarization on the collapse of
the membrane, further support the thesis.
In conclusion: it appears definitely established that true bouquet formation
is a special process, basically distinct from relic orientation, involving the opera-
tion of forces not active in the latter, and dependent on special activities of
chromosome ends, centers, and nuclear membrane. Bouquet formation while
widespread, is not, however, a universal nor essential element in meiosis. Its
SOME PROBLEMS OF MANTID MEIOSIS
adaptive significance when, as is usually the case, it occurs prior to synapsis,
lies in facilitating chromosome pairing, and was early recognized (v. Kemnitz,
1913; Gelei, 1921). The second bouquet stage, however, can have no such value
since it occurs in post-synaptic stages. It introduces a maneuvre not ordinarily
encompassed in the meiotic cycle, and one which shows the complexity of cyclical
changes which may be involved in the various constituent processes of mitosis.
Whatever its utility to the species, it emphasizes once again the amazing range
of possible variations in the interplay of different factors in normal meiotic
mechanisms.
II. Kinetochore Movements
a. Initial separation of kinetochores.
First of the several striking kinetochore movements during the meiotic
prophase is the initial separation of homologous kinetochores. The first opening
between the chromosomes of a bivalent is localized at the region of the kineto-
chores and is strongly suggestive of repulsion between these bodies. Swanson
(1942) questions the efficacy of the kinetochore as a repelling body, and holds
that the marked separation of kinetochores and the attenuation of the chromo-
some between kinetochore and nearest chiasma, usually attributed to such
repulsion, is more probably due to a poleward "pull" of the spindle. Although
this position appears well taken in Tradescantia, it is untenable in the mantids.
Here in several species, separation of homologous chromosomes is at first limited
to the kinetochore region, and occurs while the nuclear membrane is still intact.
Furthermore the plane of kinetochore separation bears no relation to the position
of the centers nor the future spindle axis.
Neither can we ascribe kinetochore separation to despiralization, although
the fact that the latter is initiated at the kinetochore region and proceeds distally
makes the assumption at first thought credible. Against it is the fact that there
is no constant correlation between time of kinetochore separation and phase of
spiralization cycle in the different species. Thus in the two geographic types of
Stagmomantis Carolina kinetochore separation starts at widely different points in
the spiralization cycle, and in Paratenodera the bivalent has reached a contraction
approximating that of metaphase before the kinetochores separate.
Again, in Callimantis (Hughes-Schrader, 1943) the differential behavior of
kinetochores in quadrivalents and in bivalents shows the degree of separation to
increase with the number of kinetochores present, other factors being equal. Of
course, in this case, some special activity of the half spindle components, inde-
pendent of the growth of the spindle as a whole, cannot be excluded, but the data
seem more simply explicable on the hypothesis of kinetochore repulsion.
Thus in mantids all available evidence points to an autonomy of the kineto-
chores in their initial separation; the force involved is localized in or operates
through the kinetochores themselves. While not establishing the nature of the
force involved, the data are in harmony with the concept of a mutual repulsion
between homologous kinetochores at certain stages of their cycle.
b. The stretch phenomenon.
A second and unique phase of kinetochore activity is initiated when, on the
breakdown of the nuclear membrane and the formation of the spindle in the
294 SALLY HUGHES-SCHRADER
nuclear area, homologous kinetochores move suddenly toward opposite poles
forcibly stretching open the bivalent between them. In bivalents with no
previous repulsion of kinetochores, their separation and bipolar orientation is
simultaneous; where repulsion precedes spindle formation, the kinetochores shift
their position so that one points toward each pole. Obviously the force involved
is operating through the kinetochores. White (1941) suggests that as the spindle
forms the kinetochores become attached to it, and the spindle then elongates
carrying the kinetochores toward the poles and stretching the chromosomes.
An analysis of spindle elongation and poleward movement of kinetochores is
given in Table I. Spindle elongation is computed from the relative distances
between the centers at the different stages; kinetochore movement similarly, in
terms of inter-kinetochore distance in the longest rod shaped bivalent. Measure-
ments are in ocular micrometer units, and each figure represents the average of
some ten measurements. A correlation between spindle elongation and kineto-
chore movement is evident; Liturgousa and the Virginia Stagmomantis, with the
greatest increase in spindle length, show the greatest kinetochore movement,
while Choeradodis with the least spindle elongation has the slightest.
Despite this, spindle elongation is definitely not the only factor in the poleward
movement of the kinetochores. In all but one of the five types the increase in
spindle length is definitely less than the distance travelled by the kinetochores
(compare columns 6 and 10 of Table I). Thus in the Virginia Stagmomantis
only 5.2 units out of 8.5 may be attributed to spindle elongation; in Choeradodis
only 2.4 units out of 5.1. The former case is particularly significant since
chromosome length, which will be a factor in interspecific differences in degree
of kinetochore separation, is the same in the two types of Stagmomantis.
Another line of evidence also demonstrates the existence of a second factor
in the poleward movement of the kinetochores. It will be noted that in each
case the elongation of the spindle follows quickly on its formation; thereafter
spindle length remains constant or even gradually decreases up to final metaphase
(Table I). The asynchrony of the bivalents in the kinetochore movement is so
great that in every case the maximum spindle length is attained before all of
the bivalents have opened. Since all bivalents are eventually stretched open,
it is clear that in many cases the poleward movement of the kinetochores is
independent of spindle elongation.
What is the nature of this second factor in the kinetochore movement?
It may be simply a continuation of the mutual repulsion of homologous kineto-
chores already apparent in some species during the preceding stage. The orien-
tation of the bivalent and the consequent direction of kinetochore movement
would then be determined by the longitudinal structure of the spindle, permitting
repulsion to act in the longitudinal axis while blocking it in any other plane.
An alternative possibility exists; an attraction between center and kinetochore
may come into play on the breakdown of the membrane and the formation of the
spindle. Such attraction is indeed suggested by the random orientation of the
kinetochores in the sex trivalent which move toward the nearer pole irrespective
of their homology. The terminal alignment of the sex chromosome, completed
before the poleward movement of the kinetochores begins, may result in too
great a separation of their kinetochores for repulsion to be effective and thus
allow the attraction between center and kinetochore to be visibly expressed.
SOME PROBLEMS OF MANTID MEIOSIS 295
It must not be forgotten, moreover, that half spindle components form between
kinetochores and center at the time these movements are taking place. Some
activity on their part, independent of the elongation of the spindle as a whole,
cannot be excluded. Thus while the nature of the forces involved cannot yet be
determined, the data do demonstrate that the poleward movement of kinetochores
and resultant stretching of the chromosomes are due to more than one agency,
and involve in addition to spindle elongation, other factors which may include
repulsion of homologous kinetochores, kinetochore-center attraction, and possibly
some activity of half spindle components.
c. Reapproach of homologous kinetochores.
On the completion of the poleward movement of the kinetochores, a precisely
opposite action ensues. Homologous kinetochores re-approach each other and
in so doing move away from the poles. The extent of this movements varies in
the different species (last column of Table I). The causal factor seems to be the
resumption of the normal coiling process, interrupted and partially undone by
the preceding stretching.
d. Metaphase orientation.
The gradual movement of the chromosomes toward the equator of the spindle
follows — and largely overlaps in time — the re-approach of the homologous kineto-
chores. It may even overlap the preceding stage of poleward kinetochore
movement. It follows from this asynchrony that some kinetochores are still
moving toward the poles while others are moving away from them, and that
movement of the whole bivalent toward the equator may be superimposed on
one or both of the others. The hypothesis that equatorial orientation is caused
by repulsion between kinetochore and center is thus either untenable or requires
the subsidiary assumption of a reversal of charge in the kinetochores — a reversal
occurring, moreover, at different times in different bivalents. Again, since the
spindle is completed long before movement of chromosomes to the equator begins,
the latter cannot be attributed to ingrowth of spindle fibers from the poles
pushing the scattered chromosomes to the equator. Finally, since both the
re-approach of homologous kinetochores and the movement to the equator occur
subsequent to the formation of half spindle components, a considerable elasticity
in the action of these elements is indicated.
During the movement to the equator the bivalents shift position as wholes
retaining their bipolar orientation. It is thus impossible to tell whether the
movement is dependent on kinetochore action, as is well established in other
forms. In the sex trivalent, however, the kinetochores definitely take the lead
in metaphase orientation. In the re-orientation of its three elements the kineto-
chores alone are active while the arms appear relaxed. As to what force or
forces underlie the movement to the equator and the co-orientation of homologous
kinetochores, the present data give no clue.
///. Chiasmata and bivalent structure
Cases already on record demonstrate that chaismata are not the sole, nor an
essential, factor in maintaining the meiotic association of homologous chromo-
somes in late prophase and metaphase (references in Hughes-Schrader, 1943).
296 SALLY HUGHES-SCHRADER
Bivalents clearly devoid of chiasmata may retain the parallel association of
their chromatids. Terminal connections between homologues at metaphase may
be formed in complete independence of previous chiasma formation. We may
take it as established that different species vary in the factors involved in the
later stages of the meiotic association of chromosomes.
The present study shows further that (1) such variation exists even among
closely related species, and (2) that bivalents within a single species may similarly
vary in the factors determining the association of their homologues. White
(1941) was unwilling on the evidence then available to admit such variation in a
basic meiotic mechanism in so closely related a group of species as the mantids.
Confronted on the one hand with the absence of chiasmata in the bivalents of
Callimantis, and on the other with contrary although indirect evidence in all
other species studied, he holds that the differences are superficial only. This
implies either that the indirect evidence for chiasmata in the majority of species
will prove unfounded or, as White further suggested, that chiasmata may be
present in the Callimantis bivalent, but, in the absence of the stretch phenomenon
in that species, are not disclosed due to the close parallel association of homologous
chromosomes up to anaphase.
As to the first alternative, the present study offers strong presumptive evidence
for the occurrence of chiasmata in certain bivalents of some species. The
evidence lies in the relatively high frequency of open cross formations in opening
bivalents, in which the association of non-sister chromatids distal to the opening
of the cross may be clearly demonstrated. That such configurations necessarily
imply previous chiasmata cannot be taken as completely proved (contrary evi-
dence in the somatic chiasmata of Drosophila ganglion cells, Kaufmann, 1934),
but the weight of evidence from many sources (heteromorphic bivalents, inter-
locking, etc.) is certainly in favor of this interpretation.
The second alternative is definitely untenable in the light of the evidence now
available. Re-investigation of Callimantis (Hughes-Schrader, 1943) has con-
firmed White's (1938) earlier conclusion that chiasmata are absent. Neither in
the parallel association of four chromatids in the prometaphase and metaphase
bivalent nor in its anaphasic disjunction are chiasmata involved. The present
study offers additional evidence that the Callimantis type of bivalent on the
one hand, and the rod and ring types of other mantids on the other, present
real differences in structure and in the forces underlying the association of the
homologues. The differences in the metaphase form of bivalents are not ascrib-
able simply to presence or absence of the stretch phenomenon. Thus we have
seen in Liturgousa one bivalent of the Callimantis type, subjected to the same
stretching that results in rod and ring formation by the other bivalents, open
out without terminal connections and on re-contraction resume the parallel
association of its homologues. Conversely, in Choeradodis, where the stretching
of the bivalents on the spindle is almost nil, rod and ring types are nevertheless
produced. In view of these lines of evidence the conclusion is justified that even
closely allied species vary in the factors underlying the late meiotic association
of homologous chromosomes.
Within the single species, also, the presence of more than one factor is demon-
strable. Thus in the case of Liturgousa cited above, factors other than chiasmata
determine the association in one bivalent, while others as consistently disclose
SOME PROBLEMS OF MANTID MEIOSIS 297
the open cross with its implication of previous chiasmata. In Choeradodis,
also, diversity of factors is evident.
Three separable factors in the late meiotic association of homologous chromo-
somes can be distinguished. They may operate separately or in various combi-
nations in bivalents of different species and in different bivalents of one species.
The first of these factors — the pairing force independent of chiasmata which
we must assume determines the persistent parallel association of chromatids in
the Callimantis type of bivalent — -I shall term, for brevity in discussion, lateral
attraction. The second, expressed in the formation of terminal connections
between separating homologues, is recognized as terminal attraction. The term
attraction is here used in a purely descriptive sense, without implication of the
nature of the force involved. The third factor is the action of chiasmata. Let
us consider these factors, as expressed in the structure of the mantid bivalents,
separately.
Lateral attraction is demonstrated most clearly in the Callimantis bivalents
where the four distinctly separated chromatids of each arm maintain their
parallel association, without chiasmata, until anaphase. It is similarly expressed
in the single Callimantis-like bivalent of Liturgousa, and in several of the Choera-
dodis bivalents whose homologous chromosomes are clearly separated but
parallelly associated until stretched apart on the spindle. The data of the
present study further demonstrate that lateral attraction may be variously
localized in the individual bivalent. This is seen in those bivalents of Choeradodis
whose homologous chromosomes show no contact during the pseudo-diakinetic
period and where chiasmata are thus excluded as a factor in the association.
Of these bivalents some show the chromosomes parallelly paired and equidistant
along their whole length. In others the pairing segment is reduced to a short
region, variously localized in different bivalents, with the homologues flaring
widely apart elsewhere. A similar differential localization of lateral attraction
is apparent in the diakinetic bivalents of the egg of the grass mite, Pediculopsis
graminum (Cooper, 1939).
Terminal attraction is expressed in the resistance of apposed chromosome
ends to the forces of anaphasic separation in the bivalents of Callimantis. In
this case no fusion or physical connection of any kind between the chromatid
ends is apparent. In the other mantid species the operation of terminal attraction
is accompanied by the formation of persistent terminal connections between the
ends of homologous chromosomes during the stretching of the bivalents on the
spindle. So extreme is the tension produced by the movement to opposite poles
of homologous kinetochores that the chromosomes are often attenuated to thin
threads, and yet the terminal connections persist. In these cases, unlike
Callimantis, a real fusion of certain constituents of the chromosome is suggested.
Terminal attraction may operate quite independently of chiasmata, as is
shown in Callimantis and in other cases on record, perhaps most strikingly in
Rhytidolomia (Schrader, 1940). This independence is also demonstrable in
Choeradodis. Here several bivalents are clearly devoid of chiasmata during the
pseudo-diakinetic period. Yet in the majority of nuclei at final metaphase all
bivalents are of either the rod or ring type, with one or two terminal connections
respectively. Obviously in certain bivalents terminal connections have formed
independently of the terminalization of chiasmata.
298 SALLY HUGHES-SCHRADER
Chiasmata are absent in late prophase and metaphase in many mantid
bivalents and even when present appear to play but a subsidiary role in meiotic
association. Since "repulsion" between pairs of chromatids, so diagnostic a
feature of diplotene and diakinesis in most forms, is not obligatory in mantids,
chiasmata when present will not be expected greatly to modify bivalent form in
prophase. Nor is the form of the metaphase bivalent dependent on chiasmata
to the extent which is usually assumed. We have seen that terminal connections
between homologous chromosomes cannot be interpreted as invariably the
sequelae of chiasma terminalization. They may also result, as is the case in
certain Choeradodis bivalents, simply from terminal attraction between those
ends which were held together earlier by lateral attraction and were finally
brought into contact by the stretching of the bivalent. When chiasmata are
present, their terminalization may well have a similar effect; it will bring chroma-
tid ends into contact and thus possibly facilitate the operation of terminal
attraction and the formation of terminal connections.
The nature of the forces involved in lateral and terminal attraction is obscure.
The formation of a non-staining pellicle common to both homologues has been
suggested, but no positive evidence of its existence is available. The intense
staining of terminal connections in many mantid bivalents suggests a fusion of
some chromosome constituent. But it must be remembered that in certain
cases of terminal attraction in other forms (e.g. Rhytidolomia, Schrader, 1940)
the chromosome ends move toward each other over a considerable distance and
any fusion that may occur is thus secondary to the initial attraction. The same
holds true for lateral attraction in the secondary metaphase pairing in spermato-
cytes of Lepidosiren (Agar, 1911), and in the re-association of previously separated
chromatids in the second meiotic division of certain coccids (Hughes-Schrader,
1931).
Somatic pairing is similar in certain respects to the lateral attraction of
meiosis. In this connection it is interesting that White (1938) finds no somatic
pairing in Callimantis in which lateral attraction is so strongly expressed. Nor
have I any convincing evidence of its occurrence in the species here reported,
with the possible exception of Choeradodis spermatogonia, and even here the
association of homologues is neither close nor constant. Oguma (1921) reports a
similarly indefinite association in the spermatogonia of Tenodera aridifolia.
Clearly there is no obligatory relation between somatic pairing and the lateral
attraction of meiosis.
SUMMARY
1. Bouquet formation. An analysis of the second polarization or bouquet
stage in the meiotic prophase of the males of several species of mantids shows
bouquet formation to be a special process, basically distinct from the relic or
Rabl orientation, and involving special activities of chromosome ends, division
centers, and nuclear membrane.
2. Kinetochore movements, (a) The initial separation or "repulsion" of the
homologous kinetochores in the meiotic bivalent is shown in several species to be
independent of division centers and spindle and appears to be autonomous to
the kinetochores. (b) The pre-metaphase poleward movement of kinetochores
and consequent stretching of the meiotic chromosomes are in part due to the
SOME PROBLEMS OF MANTID MEIOSIS
elongation of the spindle, and in part to another factor or factors which may
include kinetochore repulsion, kinetochore-center attraction, and a special activity
of the half spindle components, (c) Resumption of coiling after the pre-meta-
phase stretch results in the re-approach of the homologous kinetochores and their
movement away from the poles, (d) Movement of chromosomes to the equator
regularly overlaps the movement (c) above, and may overlap (b), thus excluding
any hypothesis of metaphase plate formation in which the chromosomes are
regarded as passive.
3. Chiasmata and bivalent structure. Three separable factors in the late
meiotic association of homologous chromosomes can be distinguished: (a) lateral
attraction, which is independent of chiasmata and is variously localized in
different bivalents; (b) terminal attraction which operates in some bivalents
quite independently of chiasmata, and in others follows chiasma terminalization;
and (c) the action of chiasmata. Absent in late prophase and metaphase in
certain bivalents, the presence of chiasmata is inferred in others from the fre-
quency of open cross configurations. These three factors may act more or less
separately, and in various combinations, even in closely allied species, and in
different bivalents of a single species.
4. Males of Stagmomantis Carolina from Virginia and from Barro Colorado
Island, C. Z., identical taxonomically and in chromosome complement, differ in
the time of spindle formation relative to the stage of bivalent development in the
meiotic prophase.
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SWANSON, C. P., 1942. Some considerations on the phenomenon of chiasma terminalization.
Amer. Nat., 76: 593-610.
WENRICH, D. H., 1916. The spermatogenesis of Phrynotettix magnus with special reference to
synapsis and the individuality of the chromosomes. Bull. Mus. Comp. Zool., Harvard
Coll., 60: 57-134.
WHITE, M. J. D., 1938. A new and anomalous type of meiosis in a mantid, Callimantis antil-
larum Saussure. Proc. Roy. Soc., 125: 516-523.
WHITE, M. J. D., 1941. The evolution of the sex chromosomes. I. The X O and XiX2Y mechan-
isms in praying mantids. Jour. Gen., 42: 143-172.
WILLIAMS, E. C., 1938. Spermatogenesis of a mantid, Choeradodis rhombicollis (Latreille).
Trans. Am. Mic. Soc., 57: 387-394.
INDEX
A CETYLCHOLINE, action of, on isolated
heart of Venus mercenaria, 79.
Amblystoma, neurulation in mechanically and
chemically inhibited, 103.
Anaphase movement, a quantitative study of,
in the aphid Tamalia, 164.
ANDERSON, THOMAS F. See Harvey and
Anderson, 151.
Annual report of the Marine Biological Labora-
tory, 1.
Aphid, production of types of, and germarial
differences, 52.
Aphid, a quantitative study of the anaphase
movement in, 164.
Arbacia punctulata, egg, rate of breaking and
size of halves of, when centrifuged in hypo-
and hypertonic sea water, 141.
Arbacia punctulata, radiosensitivity of eggs of,
in various salt solutions, 193.
Arbacia punctulata, spermatozoon and fertili-
zation membrane of, as shown by the
electron microscope, 151.
DODINE, JOSEPH HALL, AND THEODORE
NEWTON TAHMISIAN. The development
of an enzyme (tyrosinase) in the partheno-
genetic egg of the grasshopper, Melanoplus
differentialis, 157.
BUMPUS, DEAN F. See Clarke, Pierce and
Bumpus, 201.
BURT, AGNES SANXAY. Neurulation in me-
chanically and chemically inhibited Am-
blystoma, 103.
pHAETOPHORACEAE, new records of, for
° North America, 244.
Chaetosphaeridiaceae, new records of, for
North America, 244.
CLARKE, GEORGE L., E. LOWE PIERCE, AND
DEAN F. BUMPUS. The distribution and
reproduction of Sagitta elegans on Georges
Bank in relation to hydrographical condi-
tions, 201.
Crustacea, histogenesis and cyclic phenomena
of sinus gland and x-organ in, 87.
Cytoplasmic granules, the osmotic properties
of, in the sea urchin egg, 179.
F^APHNIA, analysis of population develop-
in, at different temperatures, 116.
DIPPELL, RUTH V. See Sonneborn and Dip-
pell, 36.
Distribution, of Sagitta elegans, on Georges
Bank, in relation to hydrographical condi-
tions, 201.
, Arbacia, rate of breaking and size of
halves of, when centrifuged in hypo- and
hypertonic sea water, 141.
Egg, parthenogenetic, development of tyro-
sinase in, of Melanoplus differentialis, 157.
Egg, sea urchin, the osmotic properties of cyto-
plasmic granules in, 179.
ENGLE, JAMES B. See Loosanoff and Engle, 69.
Enzyme (tyrosinase), development of, in the
parthenogenetic egg of the grasshopper,
Melanoplus differentialis, 157.
pERTILIZATION membrane, of Arbacia
punctulata, as shown by the electron
microscope, 151.
Bank, distribution and reproduc-
tion of Sagitta elegans on, in relation to
hydrographical conditions, 201.
Grasshopper, development of tyrosinase in the
parthenogenetic egg of, 157.
TT ABROBRACON, intersexuality and inter-
sexual females in, 238.
HARRIS, DANIEL L. The osmotic properties of
cytoplasmic granules of the sea urchin egg,
179.
HARVEY, ETHEL BROWNE. Rate of breaking
and size of the "halves" of the Arbacia
punctulata egg when centrifuged in hypo-
and hypertonic sea water, 141.
HARVEY, ETHEL BROWNE, AND THOMAS F.
ANDERSON. The spermatozoon and fer-
tilization membrane of Arbacia punctulata
as shown by the electron microscope, 151.
Heart, action of acetylcholine on isolated, of
Venus mercenaria, 79.
Hemolysis, osmotic, species differences in rates
of, within the genus Peromyscus, 52.
Histogenesis, and cyclic phenomena of the
sinus gland and x-organ in Crustacea, 87.
HOVANITZ, WILLIAM. Hybridization and sea-
sonal segregation in two races of a butter-
fly occurring together in two localities, 44.
301
302
INDEX
HuGHES-ScHRADER, SALLY. Polarization, ki-
netochore movements, and bivalent struc-
ture in the meiosis of male mantids, 265.
Hybridization, and seasonal segregation, in two
races of a butterfly, 44.
INHIBITION, mechanical and chemical, in
Amblystoma, 103.
Intersexuality, and intersexual females in
Habrobracon, 238.
I^INETOCHORE movements, and bivalent
structure in the meiosis of male mantids,
265.
TAWSON, CHESTER A. Germarial differ-
ences and the production of aphid types,
60.
LEVINE, HARRY P. Species differences in rates
of osmotic hemolysis within the genus
Peromyscus, 52.
Life history, of the digenetic trematode, Zo-
ogonoides laevis Linton, 227.
LOOSANOFF, VICTOR L., AND JAMES B. ENGLE.
Polydora in oysters suspended in water, 69.
V/f ANTIDS, male, polarization, kinetochore
movements, and bivalent structure, in the
meiosis of, 265.
MARINE BIOLOGICAL LABORATORY, annual re-
port of, 1.
Mating types, in variety 4, Paramecium aurelia,
36. "
Meiosis, polarization, kinetochore movements,
and bivalent structure in, of male mantids,
265.
Melanoplus differentialis, development of tyro-
sinase in the parthenogenetic egg of, 157.
Microscope, electron, spermatozoon and fer-
tilization membrane of Arbacia punctulata,
as shown by, 151.
XT EURULATION, in mechanically and chem-
ically inhibited Amblystoma, 103.
, Polydora in, suspended in water,
69.
pARAMECIUM aurelia, sexual isolation,
mating types and sexual responses to
diverse conditions in variety 4, 36.
Peromyscus, species differences in rates of
osmotic hemolysis in, 52.
PIERCE, E. LOWE. See Clarke, Pierce and
Bumpus, 201.
Polarization, kinetochore movements, and bi-
valent structure in meiosis of male man-
tids, 265.
Polydora, in oysters suspended in water, 69.
Population, analysis of development of, in
Daphnia at different temperatures, 116.
PRATT, DAVID M. Analysis of population de-
velopment in Daphnia at different tem-
peratures, 116.
PYLE, ROBERT VV. The histogenesis and cyclic
phenomena of the sinus gland and x-organ
in crustacea, 87.
OADIOSENSITIVITY, of Arbacia eggs, in
various salt solutions, 193.
RECKNAGEL, RICHARD O. See Wilbur and
Recknagel, 193.
Reproduction, of Sagitta elegans, on Georges
Bank, in relation to hydrographical condi-
tions, 201.
Ris, HANS. A quantitative study of anaphase
movement in the aphid Tamalia, 164.
C AGITTA elegans, distribution and reproduc-
tion of, on Georges Bank, 201.
Sea water, rate of breaking and size of halves
of Arbacia punctulata egg, when centri-
fuged in hypo- and hypertonic, 141.
Segregation, seasonal, and hybridization in two
races of a butterfly, 44.
Sinus gland, histogenesis and cyclic phenomena
of, in crustacea, 87.
SONNEBORN, T. M., AND RUTH V. DlPPELL.
Sexual isolation, mating types and sexual
responses to diverse conditions in variety 4,
Paramecium aurelia, 36.
Spermatozoon, of Arbacia punctulata, as shown
by the electron microscope, 151.
Structure, bivalent, in the meiosis of male
mantids, 265.
STUNKARD, HORACE W. The morphology and
life history of the digenetic trematode,
Zoogonoides laevis Linton, 1940, 227.
'T'AHMISIAN, THEODORE NEWTON. See
Bodine and Tahmisian, 157.
Tamalia, a quantitative study of the anaphase
movement in, 164.
Temperature, effect of, on population develop-
ment of Daphnia, 116.
THIVY, FRANCESCA. New records of some
marine Chaetophoraceae and Chaeto-
sphaeridiaceae for North America, 244.
Trematode, digenetic, life history and mor-
phology of, 227.
INDEX
303
Tyrosinase, the development of, in the par-
thenogenetic egg of the grasshopper, 157.
WENUS mercenaria, action of acetylcholine
on isolated heart of, 79.
ROBERT B. The action of acetyl-
choline on the isolated heart of Venus
mercenaria, 79.
WHITING, P. \Y. Intersexual females and
intersexuality in Habrobracon, 238.
WILBUR, KARL M., AND RICHARD O. RECK-
NAGEL. The radiosensitivity of eggs of
Arbacia punctulata in various salt solu-
tions, 193.
V-ORGAN, histogenesis and cyclic phe-
nomena of, in Crustacea, 87.
£OOGONOIDES laevis Linton, life history
and morphology of, 227.
Volume 85
Number 1
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
E. G. CONKLIN, Princeton University
E. N. HARVEY, Princeton University
SELIG HECHT, Columbia University
LEIGH HOADLEY, Harvard University
L. IRVING, Swarthmore College
M. H. JACOBS, University of Pennsylvania
H. S. JENNINGS, Johns Hopkins University
FRANK R. LILLIE, University of Chicago
CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
A. C. REDFIELD, Harvard University
F. SCHRADER, Columbia University
H. B. STEINBACH, Washington University
Managing Editor
AUGUST, 1943
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
SERIAL LIST
A SERIAL list of the holdings of The Marine Biological Labora-
tory was published as a separately bound supplement to the Feb-
ruary issue of The Biological Bulletin. This supplement, cov-
ering approximately 80 pages, lists with cross references the 2258
titles of journals in the Library. Titles are listed alphabetically to
conform to the arrangement of the stacks in the Library, and hence
should serve as a guide book to the Library itself, as well as an aid
in securing microfilm copies of articles. A few extra copies are
still available. Orders may be directed to The Marine Biological
Laboratory.
MICROFILM SERVICE
1 HE Library of The Marine Biological Laboratory is now pre-
pared to supply microfilms of material from periodicals included in
its extensive list. Through the generosity of Dr. Athertone Seidell,
the essential equipment has been set up and put into operation.
The Staff of The Marine Biological Laboratory Library is anxious to
extend the Microfilm Service, particularly at this time when dis-
tance makes the Library somewhat inaccessible to many who nor-
mally use it. Investigators who wish films should send to the Li-
brarian the name of the author of the paper, its title, and the name
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and year of publication. The rates are as follows: $.30 for papers
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thereof. It is hoped that many investigators will avail themselves
of this service.
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University of Pennsylvania
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LANCASTER PRESS, Inc.
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THE EXPERIENCE we have
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sixty educational publica-
tions has fitted us to meet
the standards of customers
who demand the best.
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write for estimates on journals or
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INSTRUCTIONS TO AUTHORS
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Literature cited. The list of literature cited should conform to the style set
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THE BIOLOGICAL BULLETIN
THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster
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Subscriptions and similar matter should be addressed to The Biologi-
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Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa.,
under the Act of August 24, 1912.
BIOLOGY MATERIALS
The Supply Department of the Marine Biological Labora-
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injected materials, and would be pleased to quote prices on
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PRESERVED SPECIMENS
for
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MARINE
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CONTENTS
Page
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY. . . 1
SONNEBORN, T. M., AND RUTH V. DIPPELL
Sexual Isolation, Mating Types, and Sexual Responses to
Diverse Conditions in Variety 4, Paramecium Aurelia 36
HOVANITZ, WILLIAM
Hybridization and Seasonal Segregation in Two Races of a
Butterfly Occurring Together in Two Localities 44
LEVINE, HARRY P.
Species Differences in Rates of Osmotic Hemolysis Within
the Genus Peromyscus 52
LAWSON, CHESTER A.
Germarial Differences and the Production of Aphid Types . . 60
LOOSANOFF, VICTOR L., AND JAMES B. ENGLE
Polydora in Oysters Suspended in Water 69
WAIT, ROBERT B.
The Action of Acetylcholine on the Isolated Heart of Venus
Mercenaria . 79
Volume 85
Number 2
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
E. G. CONKLIN, Princeton University
E. N. HARVEY, Princeton University
SELIG HECHT, Columbia University
LEIGH HOADLEY, Harvard University
L. IRVING, Swarthmore College
M. H. JACOBS, University of Pennsylvania
H. S. JENNINGS, Johns Hopkins University
FRANK R. LILLIE, University of Chicago
CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
A. C. REDFIELD, Harvard University
F. SCHRADER, Columbia University
H. B. STEINBACH, Washington University
Managing Editor
OCTOBER, 1943
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE &. LEMON STS.
LANCASTER, PA.
SERIAL LIST
A SERIAL list of the holdings of The Marine Biological Labora-
tory was published as a separately bound supplement to the Feb-
ruary issue of The Biological Bulletin. This supplement, cov-
ering approximately 80 pages, lists with cross references the 2258
titles of journals in the Library. Titles are listed alphabetically to
conform to the arrangement of the stacks in the Library, and hence
should serve as a guide book to the Library itself, as well as an aid
in securing microfilm copies of articles. A few extra copies are
still available. Orders may be directed to The Marine Biological
Laboratory.
MICROFILM SERVICE
1 HE Library of The Marine Biological Laboratory is now pre-
pared to supply microfilms of material from periodicals included in
its extensive list. Through the generosity of Dr. Athertone Seidell,
the essential equipment has been set up and put into operation.
The Staff of The Marine Biological Laboratory Library is anxious to
extend the Microfilm Service, particularly at this time when dis-
tance makes the Library somewhat inaccessible to many who nor-
mally use it. Investigators who wish films should send to the Li-
brarian the name of the author of the paper, its title, and the name
of the periodical in which it is printed, together with the volume
and year of publication. The rates are as follows: $.30 for papers
up to 25 pages, and $.10 for each additional 10 pages or fraction
thereof. It is hoped that many investigators will avail themselves
of this service.
Your Biological News
You would not go to the library to read the daily newspaper — probably
you have it delivered at your home to be read at your leisure. Why, then,
depend upon your library for your biological news?
Biological Abstracts is news nowadays. Abridgments of all the im-
portant biological literature are published promptly — in many cases before
the original articles are available in this country. Only by having your
own copy of Biological Abstracts to read regularly can you be sure that
you are missing none of the literature of particular interest to you. An
abstract of one article alone, which otherwise you would not have seen,
might far more than compensate you for the subscription price.
Biological Abstracts is now published in six low priced sections, as
well as the complete edition, so that the biological literature may be avail-
able to all individual biologists. Write for full information and ask for a
copy of the section covering your field.
BIOLOGICAL ABSTRACTS
University of Pennsylvania
Philadelphia, Pa.
LANCASTER PRESS, Inc.
LANCASTER, PA.
THE EXPERIENCE we have
gained from printing some
sixty educational publica-
tions has fitted us to meet
the standards of customers
who demand the best.
We shall be happy to have workers at
the MARINE BIOLOGICAL LABORATORY
write for estimates on journals or
monographs. Our prices are moderate.
INSTRUCTIONS TO AUTHORS
The Biological Bulletin accepts papers on a variety of subjects of biologi-
cal interest. In general, a paper will appear within three months of the date of
its acceptance. The Editorial Board requests that manuscripts conform to the
requirements set below.
Manuscripts. Manuscripts should be typed in double or triple spacing on
one side of paper, 8l/2 by 11 inches.
Tables should be typewritten on separate sheets and placed in correct
sequence in the text. Explanations of figures should be typed on a separate
sheet and placed at the end of the text. Footnotes, numbered consecutively,
may be placed on a separate sheet at the end of the paper.
A condensed title or running page head of not more than thirty-five letters
should be included.
Manuscripts must be returned to the Editor with the galley proof. Page
proofs will be sent only on request.
Figures. The dimensions of the printed page, 5 by 7% inches, should be
kept in mind in preparing figures for publication. Illustrations should be large
enough so that all details will be clear after appropriate reduction. Explana-
tory matter should be included in legends as far as possible, not lettered on the
illustrations. Figures should be prepared for reproduction as line cuts or half-
tones; other methods will be used only at the author's expense. Figures to be
reproduced as line cuts should be drawn in black ink on white paper or blue-
lined co-ordinate paper; those to be reproduced as halftones should be mounted
on Bristol board and any designating letters or numbers should be made di-
rectly on the figures. The author's name should appear on the reverse side of
all figures.
Literature cited. The list of literature cited should conform to the style set
in this issue of The Biological Bulletin. Papers referred to in the manuscript
should be listed on separate pages headed "Literature Cited." Where there are
several papers cited, by the same author, the author's name should be repeated
in each case.
Mailing. Manuscripts should be packed flat, not folded or rolled. Large
charts and graphs may be rolled in a mailing tube.
Reprints. Authors will be furnished, free of charge, one hundred reprints
without covers. Additional copies may be obtained at cost; approximate
figures will be furnished upon request.
THE BIOLOGICAL BULLETIN
THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster
Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania.
Subscriptions and similar matter should be addressed to The Biologi-
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts.
Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers,
$1.75. Subscription per volume (three issues), $4.50.
Communications relative to manuscripts should be sent to the Manag-
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts,
between July 1 and October 1, and to the Department of Zoology, Wash-
ington University, St. Louis, Missouri, during the remainder of the year.
Entered as second-class matter May 17, 1930, at the post office at Lancaster. Pa.,
under the Act of August 24, 1912.
BIOLOGY MATERIALS
The Supply Department of the Marine Biological Labora-
tory has a complete stock of excellent plain preserved and
injected materials, and would be pleased to quote prices on
school needs.
PRESERVED SPECIMENS
for
Zoology, Botany, Embryology,
and Comparative Anatomy
LIVING SPECIMENS
for
Zoology and Botany
including Protozoan and
Drosophila Cultures, and
Animals for Experimental and
Laboratory Use.
MICROSCOPE SLIDES
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Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.
CATALOGUES SENT ON REQUEST
Supply Department
MARINE
BIOLOGICAL LABORATORY
Woods Hole, Massachusetts
CONTENTS
Page
PYLE, ROBERT W.
The Histogenesis and Cyclic Phenomena of the Sinus Gland
and X-Organ in Crustacea 87
BURT, AGNES SANXAY
Neurulation in Mechanically and Chemically Inhibited
Amblystoma 103
PRATT, DAVID M.
Analysis of Population Development in Daphnia at Different
Temperatures 116
HARVEY, ETHEL BROWNE
Rate of Breaking and Size of the "Halves" of the Arbacia
Punctulata Egg when Centrifuged in Hypo- and Hypertonic
Sea Water 141
HARVEY, ETHEL BROWNE, AND THOMAS F. ANDERSON
The Spermatozoon and Fertilization Membrane of Arbacia
Punctulata as Shown by the Electron Microscope 151
BODINE, JOSEPH HALL, AND THEODORE NEWTON TAHMISIAN
The Development of an Enzyme (Tyrosinase) in the Par-
thenogenetic Egg of the Grasshopper, Melanoplus Differen-
tialis 157
RIS, HANS
A Quantitative Study of Anaphase Movement in the Aphid
Tamalia . 164
Volume 85 Number 3
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
E. G. CONKLIN, Princeton University FRANK R. LlLLlE, University of Chicago
E. N. HARVEY, Princeton University CARL R. MOORE, University of Chicago
SELIG HECHT, Columbia University GEORGE T. MOORE, Missouri Botanical Garden
LEIGH HOADLEY, Harvard University T. H. MORGAN, California Institute of Technology
L. IRVING, Swarthmore College G. H. PARKER, Harvard University
M. H. JACOBS, University of Pennsylvania A. C. REDFIELD, Harvard University
H. S. JENNINGS, Johns Hopkins University F. SCHRADER, Columbia University
H. B. STEINBACH, Washington University
Managing Editor
DECEMBER, 1943
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE 8C LEMON STS.
LANCASTER, PA.
SERIAL LIST
A SERIAL list of the holdings of The Marine Biological Labora-
tory was published as a separately bound supplement to the Feb-
ruary issue of The Biological Bulletin. This supplement, cov-
ering approximately 80 pages, lists with cross references the 2258
titles of journals in the Library. Titles are listed alphabetically to
conform to the arrangement of the stacks in the Library, and hence
should serve as a guide book to the Library itself, as well as an aid
in securing microfilm copies of articles. A few extra copies are
still available. Orders may be directed to The Marine Biological
Laboratory.
MICROFILM SERVICE
1 HE Library of The Marine Biological Laboratory is now pre-
pared to supply microfilms of material from periodicals included in
its extensive list. Through the generosity of Dr. Athertone Seidell,
the essential equipment has been set up and put into operation.
The Staff of The Marine Biological Laboratory Library is anxious to
extend the Microfilm Service, particularly at this time when dis-
tance makes the Library somewhat inaccessible to many who nor-
mally use it. Investigators who wish films should send to the Li-
brarian the name of the author of the paper, its title, and the name
of the periodical in which it is printed, together with the volume
and year of publication. The rates are as follows: $.30 for papers
up to 25 pages, and $.10 for each additional 10 pages or fraction
thereof. It is hoped that many investigators will avail themselves
of this service.
Your Biological News
You would not go to the library to read the daily newspaper — probably
you have it delivered at your home to be read at your leisure. Why, then,
depend upon your library for your biological news ?
Biological Abstracts is news nowadays. Abridgments of all the im-
portant biological literature are published promptly — in many cases before
the original articles are available in this country. Only by having your
own copy of Biological Abstracts to read regularly can you be sure that
you are missing none of the literature of particular interest to you. An
abstract of one article alone, which otherwise you would not have seen,
might far more than compensate you for the subscription price.
Biological Abstracts is now published in six low priced sections, as
well as the complete edition, so that the biological literature may be avail-
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BIOLOGICAL ABSTRACTS
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INSTRUCTIONS TO AUTHORS
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THE BIOLOGICAL BULLETIN
THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster
Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania.
Subscriptions and similar matter should be addressed to The Biologi-
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Entered as second-class matter May 17, 1930, at the post office at Lancaster. Pa.,
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CONTENTS
Page
HARRIS, DANIEL L.
The Osmotic Properties of Cytoplasmic Granules of the Sea
Urchin Egg 179
WILBUR, KARL M., AND RICHARD O. RECKNAGEL
The Radiosensitivity of Eggs of Arbacia Punctulata in Various
Salt Solutions 193
CLARKE, GEORGE L., E. LOWE PIERCE AND DEAN F. BUMPUS
The Distribution and Reproduction of Sagitta Elegans on
Georges Bank in Relation to the Hydrographical Conditions . 201
STUNKARD, HORACE W.
. The Morphology and Life History of the Digenetic Trema-
tode, Zoogonoides Laevis Linton, 1940 227
WHITING, P. W.
Intersexual Females and Intersexuality in Habrobracon .... 238
THIVY, FRANCESCA
New Records of Some Marine Chaetophoraceae and Chaeto-
sphaeridiaceae for North America 244
HUGHES-SCHRADER, SALLY
Polarization, Kinetochore Movements, and Bivalent Struc-
ture in the Meiosis of Male Mantids . 265
r
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